pPIC6 Vector - Thermo Fisher Scientific
Contents
1. 1 liter dissolve 6 g sodium phosphate monobasic 372 mg EDTA 50 mL glycerol in 900 mL deionized water 3 Use NaOH to adjust pH to 7 4 and bring up the volume to 1 liter Store at 4 C 4 Add protease inhibitors immediately before use 20 Blasticidin Description Handling Blasticidin Preparing and Storing Stock Solutions 21 Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells Takeuchi et al 1958 Yamaguchi et al 1965 Resistance is conferred by expression of either one of two blasticidin S deaminase genes BSD from Aspergillus terreus Kimura et al 1994 or bsr from Bacillus cereus Izumi et al 1991 These deaminases convert blasticidin S to a non toxic deaminohydroxy derivative Izumi et al 1991 Always wear gloves mask goggles and protective clothing e g a laboratory coat when handling blasticidin Weigh out blasticidin and prepare solutions ina hood Blasticidin is soluble in water Water is generally used to prepare stock solutions of 5 to 10 mg mL e Dissolve blasticidin in sterile water and filter sterilize the solution e Aliquot in small volumes suitable for one time use see last point below and freeze at 20 C for long term storage or store at 4 C for short term storage e Aqueous stock solutions are stable for 1 2 weeks at 4 C and 6 8 weeks at 20 C e2. ACAACTAATT Sfu EcoR Pml Sfi Asp718 Kpn Xho I I I 1 I 1 I ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC Sac Il Not SnaB c myc epitope l l l GGCGGCCGCC AGCTT ACGTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Polyhistidine tag l l AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA Asn Ser Ala Val Asp His His His His His His CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA 3 AOX1 priming site GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT 3 polyadenylation site TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC Continued on next page Cloning into pPIC6 A B and C Continued E coli Transformation Important Preparing a Glycerol Stock Plasmid Preparation Transform your ligation mixtures into a competent recA endA E coli strain e g TOP10 DH5a JM109 and select on Low Salt LB agar plates containing 100 ug mL blasticidin see below Note that there is no blue white screening for the presence of insert with pPIC6 A B or C Once you have obtained blasticidin resistant colonies pick 10 transformants and screen for the presence and orientation of your insert To facilitate selection of blasticidin resistant E coli the salt concentration of the medium must remain low lt 90 mM and the pH must be 7 0 Prepare Low Salt LB broth and pla
3. Pml Sfi I I I 1 ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC Asp718 Kpn I Xho I 1 I GGATCGGTAC CTCGAGCCGC Sac Il Not Xba c myc epitope i GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Polyhistidine tag l AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT Asn Ser Ala Val Asp His His His His His His CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG 3 AOX1 priming site GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT 3 polyadenylation site TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TGA GTTTGTAGCC TTAGACATGA kkk TCTTGCTAGA TTCTAATCAA CATTTTTGAT ACTTTTTTAT TTC Continued on next page Cloning into pPIC6 A B and C Continued Multiple Cloning Below is the multiple cloning site for pPIC6 C Restriction sites are labeled to Site of pPIC6 C indicate the cleavage site The boxed nucleotides indicate the variable region The 811 871 931 991 1041 1097 1157 L217 multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of pPIC6 C is available for downloading from www invitrogen com or from Technical Support see page 35 For a map and a description of the features of pPIC6 refer to the Appendix pages 16 17 5 end of AOX1 mRNA 5 AOX7 priming site T AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TIGCGACTGG TTCCAATTGA sel CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA
4. Small scale expression conditions may not be optimal for your protein For this reason the method you choose for detection e g SDS PAGE Western or functional assay may be an important factor in determining the success of expression If your method of detection does not reveal any expression you may want to consider using a more sensitive method Once a positive clone has been identified large scale expression can be carried out in shake flask or fermentation and expression conditions can be optimized Once you have obtained blasticidin resistant transformants it is not necessary to maintain your recombinant Pichia clone in medium containing blasticidin for expression studies Blasticidin is only required for initial screening and selection of recombinant clones We recommend that you use the following techniques to assay expression of your protein Note that the c myc epitope and the polyhistidine 6xHis tag will contribute 2 5 kDa to the size of your protein Be sure to account for any additional amino acids that are in between the end of your native protein and the c myc epitope Technique Method of Detection Sensitivity SDS PAGE Visualization by eye Can detect as little as Coomassie stained 100 ng in a single band SDS PAGE Visualization by eye Can detect as little as Silver stained 2 ng in a single band Western Analysis Antibody to your particular Can detect as little as protein 1 10 pg depending on Anti
5. a commercial license Access to the Expression Kit and Vector must be limited solely to those officers employees and students of your institution who need access to perform the above described research or evaluation You must inform each such officer em ployee and student of the provisions of this license agreement and require them to agree in writing to be bound by the provisions of this license agreement You may not distribute any Expression Vector or host strain contained herein or in the Expression Kit to others even those within your own institution You may only transfer modified altered or original material from the Expression Kit or Vector to a third party following written notification of and written approval from Life Technologies so that the recipient can be licensed You may not assign sub license rent lease or otherwise transfer this license agreement or any of the rights or obligation there under except as expressly permitted by Life Technologies and RCT This license agreement is effective until terminated You may terminate it at any time by destroying all Pichia Expression products in your control It will also terminate automatically if you fail to comply with the terms and conditions of the license agreement You shall upon termination of the license agreement destroy all Pichia Expression products in your control and so notify Life Technologies in writing You may contact Research Corporation Technologies at the f
6. basic steps needed to clone and express your gene of interest in pPIC6 Step Action Page 1 Propagate pPIC6 A B and C by transformation into a recA 2 endAl E coli strain such as TOP10 DH5a or JM109 2 Develop a cloning strategy and ligate your gene into one of 3 6 the pPIC6 vectors in frame with the C terminal tag 3 Transform into E coli and select transformants on Low Salt LB 7 plates containing 100 g mL blasticidin 4 Analyze 10 20 transformants by restriction mapping or 7 sequencing to confirm in frame fusion of your gene with the C terminal tag 5 Purify and linearize the recombinant plasmid for 7 10 transformation into Pichia pastoris 6 Transform X 33 or your Pichia strain and plate onto YPDS 10 11 plates containing the appropriate concentration of blasticidin Select for blasticidin resistant transformants 10 11 Optimize expression of your gene 12 13 Purify your fusion protein on metal chelating resin e g 14 15 ProBond Methods Cloning into pPIC6 A B and C General Molecular Biology Techniques E coli Strain Transformation Method Maintaining Plasmids For assistance with E coli transformations restriction enzyme analysis DNA biochemistry and plasmid preparation refer to Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 Many E coli strains
7. for blasticidin resistance in Pichia and may be used to optimize expression and purification conditions for your host strain The pPIC6 lacZ plasmid expresses B galactosidase fused at the C terminus to the c myc epitope and the polyhistidine tag see page 18 for more information Expression of the 119 kDa fusion protein is driven by the Paoxi promoter and is inducible with methanol The fusion protein is visible on a Coomassie stained SDS polyacrylamide gel and can be detected using the Anti myc antibodies see page 34 or using an ONPG assay P Gal Assay Kit see page 33 You will need the following reagents for transforming Pichia and selecting transformants on blasticidin Note Inclusion of sorbitol in YPD plates stabilizes electroporated cells as they appear to be somewhat osmotically sensitive e 5 10 ug pure pPIC6 plasmid containing your insert e YPD Medium e 50 mL conical polypropylene tubes e 1 liter cold 4 C sterile water place on ice the day of the experiment e 25 mL cold 4 C sterile 1 M sorbitol place on ice the day of the experiment e 30 C incubator e Electroporation device and 0 2 cm cuvettes e YPDS plates containing the appropriate concentration of blasticidin see page 20 for recipe To promote integration we recommend that you linearize your pPIC6 construct within the 5 AOX1 region The table below lists unique sites that may be used to linearize pPIC6 prior to transformation Other restriction site
8. l in pPIC6 A AOX1 transcription termination region bases 1078 1419 Xba in pPIC6 B TEF1 promoter bases 1420 1828 SnaB in pPIC6 C EM7 promoter bases 1833 1899 Blasticidin resistance gene bases 1900 2298 CYC7 transcription termination region bases 2327 2644 pUC origin bases 2655 3328 complementary strand Continued on next page 16 pPIC6 Vector Continued Features of pPIC6 pPIC6 A 3382 bp pPIC6 B 3380 bp and pPIC6 C 3381 bp contain the A B and C 17 following elements All features have been functionally tested Feature Benefit 5 AOX1 promoter A 942 bp fragment containing the AOX1 promoter that allows methanol inducible high level expression of the gene of interest in Pichia Targets plasmid integration to the AOX1 locus Multiple cloning site Allows insertion of your gene into the expression vector c myc epitope Glu Gln Lys Leu Ie Ser Glu Glu Asp Leu Allows detection of your recombinant fusion protein with the Anti myc Antibodies Evans et al 1985 C terminal polyhistidine 6xHis tag Allows purification of your recombinant fusion protein on metal chelating resin such as ProBond In addition the C terminal polyhistidine tag is the epitope for the Anti His C term Antibodies AOX1 transcription termination TT region Native transcription termination and polyadenylation signal from AOX1 gene 260 bp that permits efficient 3 mRNA
9. myc antibodies see the detection method next page alkaline phosphatase Anti His C term antibodies horseradish peroxidase see the next page radiolabeled antibody Functional assay Varies depending on assay Varies depending on assay Used to compare relative amounts of protein Continued on next page 12 Expression in Pichia Continued Polyacrylamide Gel Electrophoresis Western Analysis Control Strain Important Expression Guidelines 13 To facilitate separation and visualization of your recombinant protein by polyacrylamide gel electrophoresis a wide range of pre cast NuPAGE and Tris Glycine polyacrylamide gels are available from Invitrogen In addition Invitrogen also carries a large selection of molecular weight protein standards and staining kits For more information about the appropriate gels standards and stains to use to visualize your recombinant protein visit www invitrogen com or call Technical Support see page 35 To detect expression of your recombinant fusion protein by western blot analysis you may use the Anti myc antibodies or the Anti His C term antibodies available from Invitrogen see page 34 for ordering information or an antibody to your protein of interest In addition the Positope Control Protein is available from Invitrogen for use as a positive control for detection of fusion proteins containing a c myc epitope or a polyhistidine 6xHis t
10. page 34 for ordering information The Pichia EasyComp Transformation Kit provides reagents to prepare 6 preparations of competent cells Each preparation yields enough competent cells for 20 transformations Competent cells may be used immediately or frozen and stored for future use For more information visit www invitrogen com or contact Technical Support page 35 The pPIC6 vectors do not contain a yeast origin of replication Transformants can only be isolated if recombination occurs between the plasmid and the Pichia genome Since pPIC6 does not contain the HIS4 gene integration can only occur at the AOXI locus Vector linearized within the 5 AOX1 region will integrate by gene insertion into the host 5 AOX1 region Therefore the Pichia host that you use will determine whether the recombinant strain is able to metabolize methanol Mut or not Mut To generate a Mut recombinant strain you must use a Pichia host that contains the native AOX1 gene e g X 33 SMD1168H If you wish to generate a Mut recombinant strain then use a Pichia host that has a disrupted AOX1 gene e g KM71H Note The X 33 strain supplied with the pPIC6 vector contains the native AOX1 gene therefore the recombinant strain will be Mut Continued on next page Pichia Transformation Continued Positive Control Before Starting Linearizing Your pPIC6 Construct The pPIC6 lacZ plasmid is provided as a positive control vector
11. 0 email outlicensing invitrogen com The Pichia Expression System is based on the yeast Pichia pastoris Pichia pastoris was developed into an expression system by scientists at Salk Institute Biotechnology Industry Associates SIBIA and Phillips Petroleum for high level expression of recombinant proteins All patents for Pichia pastoris and licenses for its use as an expression system are owned by Research Corporation Technologies RCT Inc Tucson Arizona Life Technologies has an exclusive li cense to sell Pichia expression kits and vectors to scientists for research purposes only under the terms described below Use of Pichia pastoris by commercial entities for any commercial purpose requires the user to obtain a commercial license as detailed below Before using any Pichia expression product please read the following license agreement If you do not agree to be bound by its terms contact Life Technologies within 10 days for authorization to return the unused Pichia expression products and to receive a full refund If you do agree to the terms of this license agreement please complete the User Registration Card and return it to Life Technologies before using the product Life Technologies Corporation Life Technologies grants you a non exclusive license to use the enclosed Pichia expression vectors Expression Vector for academic research or for evaluation purposes only The Expression Vectors are being transferred to yo
12. 00 x g to pellet Resuspend the pellet in 1 mL of YPD and incubate at 30 C with shaking After 1 hour and 4 hours plate 25 to 100 uL on YPD plates containing the appropriate concentration of blasticidin Incubate the plates for 2 3 days at 30 C 24 Constructing n Vitro Multimers Experimental At this point you should have your gene cloned into the multiple cloning site of Outline pPIC6 A B or C To generate multiple copies of your expression cassette Step Description 1 Digest pPIC6 containing your gene of interest with Bgl II and BamH I to release the expression cassette Paox plus your gene 2 To clone multiple copies of the expression cassette linearize pPIC6 containing your gene of interest using BamH I Note that the BamH I linearized vector already contains one copy of your expression cassette 3 Treat the Bgl II BamH I expression cassette with ligase in vitro Note that Bgl II and BamH I share 4 bases in common between their recognition sites GATO 4 Generate head to tail head to head and tail to tail multimers Head to tail ligation which is the correct orientation for expression will destroy both the BamH I and Bgl II sites 5 Treat the ligation mix with BamH I and Bgl II to eliminate head to head and tail to tail multimers Ligate into BamH I linearized recombinant pPIC6 Transform into E coli and analyze recombinant plasmids for copy number
13. 92 10 Take your BamH I digest from Digesting Recombinant pPIC6 Step 2 and phenol extract then ethanol precipitate the DNA Resuspend in 17 pL of sterile water Set up a 20 uL dephosphorylation reaction in a microcentrifuge tube as follows BamH I digested recombinant pPIC6 page 26 Step 2 17 uL 10X CIAP Buffer 2 uL CIAP 1 Unit uL 1 uL Incubate at 37 C for 15 minutes Add 30 uL of sterile water to the reaction for a final volume of 50 uL Add 50 uL of phenol chloroform and extract your DNA solution Precipitate the DNA by adding 5 uL of 3 M sodium acetate and 110 uL of 100 ethanol Incubate on ice for 30 minutes Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4 C Carefully decant the supernatant Wash the nucleic acid pellet with 80 ethanol centrifuge 2 minutes and remove the ethanol Centrifuge again for 1 minute remove residual ethanol and air dry the pellet Resuspend pellet in 8 uL sterile water Save on ice if you plan to ligate your insert immediately see Ligation and Digestion of Expression Cassette or store at 20 C Ligation of the expression cassette will generate head to tail head to head and tail to tail multimers Creation of head to tail multimers will be in the correct orientation for expression and will destroy both the BamH I and Bgl II sites between the expression cassettes Digestion of the multimers with BamH I and Bgl II will eliminate those multimers with tai
14. Analysis of Purification Scale up 15 For sample application onto ProBond you need Native Binding Buffer pH 7 8 and a 2 mL ProBond column pre equilibrated using native conditions 1 Combine 1 mL 2 3 mg mL total protein of Pichia lysate with 7 mL Native Binding Buffer 2 Take a pre equilibrated ProBond column and resuspend the resin in 4 mL of the diluted lysate from Step 1 3 Seal the column and batch bind by rocking gently at room temperature for 10 minutes 4 Let the resin settle by gravity or low speed centrifugation 800 x g and carefully remove the supernatant Save the supernatant to check for unbound protein 5 Repeat Steps 2 through 4 with the remaining 4 mL of diluted lysate Proceed to Column Washing and Elution Under Native Conditions in the ProBond Purification manual Use the recommendations noted for bacterial cell lysates Use the protocol above except pre equilibrate the ProBond column using Denaturing Binding Buffer and combine 1 mL of the Pichia cell lysate with 7 mL of the Denaturing Binding Buffer We have observed that some Pichia proteins may be retained on the ProBond column using native purification conditions Optimization of the purification see ProBond Purification manual or using denaturing purification may remove these non specific Pichia proteins Be sure to save all fractions washes and flow through for analysis by SDS PAGE You may need to use wes
15. C CTCGAGCCGC c myc epitope T GGCGGCCGCC AGCTT GGGCCC GAA CAA AAA CTC Glu Gln Lys Leu 1 ATC TCA GAA GAG GAT CTG Ile Ser Glu Glu Asp Leu Polyhistidine tag i AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT Asn Ser Ala Val Asp His His His His His 1 CAT TGA GTTTGTAGCC TTAGACATGA His KKK CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA 3 AOX1 priming site j 1 GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT 3 polyadenylation site l TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC Continued on next page Cloning into pPIC6 A B and C Continued Multiple Cloning Site of pPIC6 B 811 871 931 991 1040 1096 1156 1216 l l GGCGGCCGCC AGCTT TCTA Below is the multiple cloning site for pPIC6 B Restriction sites are labeled to indicate the cleavage site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of pPIC6 B is available for downloading from www invitrogen com or from Technical Support see page 35 For a map and a description of the features of pPIC6 refer to the Appendix pages 16 17 5 end of AOX7 mRNA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA 5 AOX1 priming site TTGCGACTGG TTCCAATTGA EOE CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT Sfu EcoR I
16. EEE RENESA 35 Purchaser Notification wx sick sae ecole od ATT E EE A E ARA 36 TEA E EN PN EE IA E US EEA AEO E E A T 38 iii Kit Contents and Storage Shipping and Storage Kit Contents X 33 Pichia Strain Reference Sources Intended Use pPIC6 vectors are shipped on wet ice Upon receipt store vectors at 20 C and store the X 33 stab at 4 C The kit contents are listed below Item Composition Amount pPIC6 A Band C 40 uL of 0 5 ug L vector in 10 mM Tris HCl 20 ug 1 mM EDTA pH 8 0 pPIC6 lacZ 40 uL of 0 5 ug uL vector in 10 mM Tris HCI 20 ug 1 mM EDTA pH 8 0 X 33 Pichia strain 1 stab in YPD medium 1 stab The X 33 Pichia strain has the following genotype and phenotype Genotype Wild type Phenotype Mut For long term storage of your Pichia strain stab we recommend preparing a glycerol stock immediately upon receipt and storing at 80 C TM The pPIC6 A B and C vectors may be used with the EasySelect Pichia Expression Kit or the Original Pichia Expression Kit available from Invitrogen see page 34 for ordering Additional information about recombinant protein expression in Pichia pastoris is provided in the manuals for the EasySelect Pichia Expression Kit and the Original Pichia Expression Kit The manuals can be downloaded from www invitrogen com or obtained by contacting Technical Support see page 35 More detailed information and protocols dealing with
17. L Total volume 10 uL Incubate overnight at 16 C You may store the ligation reactions at 20 C until ready to use or transform 1 10 uL of each ligation mix into competent E coli Note that the amount of the ligation mixture you transform depends on whether you use electrocompetent or chemically competent cells You may have to decrease the amount you to transform into electrocompetent cells to prevent arcing Remember to include the vector only and cells only controls to evaluate your experiment The vector only will indicate whether your vector was dephosphorylated Since the CIAP reaction is not 100 and because you often get degradation of the ends there might be a few colonies on this plate The cells only plate should have no colonies at all 1 2 Transform competent E coli by your method of choice After adding medium to the transformed cells and allowing them to recover plate 10 uL and 100 uL of each transformation mix onto Low Salt LB plates containing 100 g mL blasticidin Save the remainder of your transformation mix at 4 C Incubate overnight at 37 C If you do not get transformants or very few transformants plate out the remainder of the transformation mix onto Low Salt LB blasticidin plates Continued on next page 30 Constructing n Vitro Multimers Continued Analyzing Transformants 31 Pick 20 transformants and inoculate each colony into 2 mL Low Salt LB containing 100 pg mL bla
18. L YPD medium to each tube Shake 200 rpm the cultures at 30 C After 1 hour take one of the tubes and plate out all of the cells by spreading 200 uL on 150 mm plates containing the appropriate concentration of blasticidin 5 Optional Continue incubating the other culture for three more hours for a total of four hours and then plate out all of the cells by spreading 200 uL on 150 mm plates containing the appropriate concentration of blasticidin 6 Incubate plates for 2 to 4 days at 30 C until colonies form If you used a Pichia strain containing a native AOX1 gene e g X 33 GS115 SDM1168H as the host for your pPIC6 construct your blasticidin resistant transformants will be Mut If you used a strain containing a deletion in the AOX1 gene e g KM71H your transformants will be Mut If you wish to verify the Mut phenotype of your blasticidin resistant transformants refer to the general guidelines provided in the EasySelect Pichia Expression Kit manual or the Original Pichia Expression Kit manual or to published reference sources Higgins and Cregg 1998 You are now ready to test your transformants for expression of your gene of interest Proceed to Expression in Pichia next page Expression in Pichia Introduction Note Detecting Recombinant Proteins in Pichia The primary purpose of small scale expression is to identify confirm a recombinant Pichia clone that is expressing the correct protein
19. Mut Phenotype 11 When selecting for blasticidin resistant transformants we often observe colonies of two different sizes large and small on YPD plates containing 300 ug mL blasticidin Generally large colonies represent transformants containing pPIC6 integrants while small colonies represent transformants containing pPIC6 non integrants These non integrants have transduced the pPIC6 plasmid and therefore exhibit a low level of blasticidin resistance in the initial selection process Upon subsequent screening these non integrant transformants do not retain blasticidin resistance When choosing a blasticidin resistant transformant for your expression studies we recommend that you pick blasticidin resistant colonies from the initial transformation plate and streak them on a second YPD plate containing the appropriate concentration of blasticidin Select transformants which remain blasticidin resistant for further studies Generally several hundred blasticidin resistant colonies are generated using the protocol on the previous page If more colonies are needed the protocol may be modified as described below Note that you will need 20 150 mm plates with YPDS agar containing the appropriate concentration of blasticidin 1 Set up two transformations per construct and follow Steps 1 through 5 of the Transformation by Electroporation protocol previous page 2 After 1 hour in 1 M sorbitol at 30 C Step 5 previous page add 1 m
20. Pichia pastoris may also be found in the following general reference see page 34 for ordering information Higgins D R and Cregg J M 1998 Pichia Protocols In Methods in Molecular Biology Vol 103 J M Walker ed Humana Press Totowa NJ For research use only Not intended for any animal or human therapeutic or diagnostic use Continued on next page Kit Contents and Storage Continued Materials Supplied by the User For the procedures described in this manual you will need the following reagents and equipment Additional reagents may be required Check each experiment to ensure you have all the reagents necessary Equipment Microbiological equipment Electroporation device and 0 2 cm cuvettes or reagents for transformation 16 C 37 C and 65 C water baths or temperature blocks 30 C and 37 C shaking and non shaking incubators Hemocytometer or Countess Automated Cell Counter see page 33 Microtiter plates optional Reagents Electrocompetent or chemically competent E coli must be recA endA for transformation see page 33 Restriction enzymes and appropriate buffers Agarose and low melt agarose S N A P Gel Purification Kit or glass milk Sterile water CIAP calf intestinal alkaline phosphatase 1 unit pL 10X CIAP Buffer Phenol chloroform 3 M sodium acetate 100 ethanol 80 ethanol T4 Ligase 2 5 units uL 10X Ligation Buffer with ATP Low Salt LB medium see pag
21. a Expression Kit manual Purification Introduction Metal Chelating Resin Binding Capacity of ProBond Important Preparing Cell Lysates In this section you will grow and induce a 10 200 mL culture of your Pichia transformant for trial purification on a metal chelating resin such as ProBond or Ni NTA You may harvest the cells and store them at 80 C until you are ready to purify your fusion protein or you may proceed directly with protein purification Note that this section only describes preparation of cell lysates and sample application onto ProBond For instructions on how to prepare and use ProBond resin refer to the ProBond Purification manual TM You may use the ProBond Purification System or Ni NTA Purification System or a similar product to purify your 6xHis tagged protein see page 33 for ordering Both purification systems contain a metal chelating resin specifically designed to purify 6xHis tagged proteins Before starting be sure to consult the ProBond Purification System manual or Ni NTA Purification System manual to familiarize yourself with the buffers and the binding and elution conditions If you are using another resin consult the manufacturer s instructions TM One milliliter of ProBond resin binds from 1 5 mg of recombinant protein This amount can vary depending on the protein Throughout the following protocol be sure to keep the cell lysate and fractio
22. ag WesternBreeze Chromogenic Kits and WesternBreeze Chemiluminescent Kits are available from Invitrogen to facilitate detection of antibodies by colorimetric or chemiluminescent methods see page 33 for ordering For more information visit www invitrogen com or call Technical Support see page 35 If you have transformed the pPIC6 lacZ plasmid into your Pichia host strain you may use this recombinant strain as a positive control for expression Expression of B galactosidase in shake flasks is detectable after 48 hours and reaches the maximum at 96 hours P galactosidase may be detected using SDS PAGE and staining the gel with Coomassie Blue or by ONPG assay P Gal Assay Kit see page 33 Cells expressing P galactosidase can be detected by plating on medium containing methanol and X gal TM For a small scale Mut expression protocol refer to the EasySelect Pichia Expression Kit manual or to general reference texts Because the pPIC6 vector does not contain the HIS4 gene his4 Pichia strains containing the integrated plasmid must be grown in medium containing 0 004 histidine If histidine is not present in the medium the cells will not grow If you used X 33 as the host strain supplementation of the medium with histidine is not required General guidelines to perform small scale expression optimize expression and scale up of expression are provided in the EasySelect Pichia Expression Kit manual or the Original Pichi
23. alification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or document
24. are suitable for the propagation of the pPIC6 vectors including TOP10 JM109 and DH5a We recommend that you propagate the pPIC6 vectors in E coli strains that are recombination deficient recA and endonuclease A deficient endA For your convenience TOP10 E coli are available as chemically competent or electrocompetent cells from Invitrogen see page 33 You may use any method of choice for transformation Chemical transformation is the most convenient for many researchers Electroporation is the most efficient and the method of choice for large plasmids The pPIC6 and pPIC6 lacZ vectors contain the blasticidin resistance gene to allow selection of the plasmid using blasticidin To propagate and maintain the pPIC6 and pPIC6 lacZ plasmids we recommend using the following procedure 1 Use a small amount of the supplied plasmid stock solution in TE pH 8 0 to transform a recA endA E coli strain like TOP10 DH5a JM109 or equivalent 2 Select transformants on Low Salt LB plates containing 100 ug mL blasticidin see page 19 for a recipe 3 Prepare a glycerol stock from each transformant containing plasmid for long term storage see page 7 Continued on next page Cloning into pPIC6 A B and C Continued General Considerations Cloning Considerations Constructing Multimeric Plasmids The following are some general points to consider when using pPIC6 to express your gene of interest in Pichia The cod
25. ation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 51 Blasticidin and the Blasticidin Selection Marker Limited Use Label License No 74 Pichia pastoris Expression System This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany Blasticidin and the blasticidin resistance gene bsd are the subject of U S Patent No 5 527 701 sold under patent license for research purposes only For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 650
26. by digesting with Bgl II and BamH I Alternative You may wish to build each desired multimer in increments by ligating each Procedure additional expression cassette one or two at a time into pPIC6 A B or C For example Step Description 1 Digest pPIC6 containing one copy of your gene with BamH I 2 Ligate a single copy of the Bg II BamH I expression cassette into BamH I digested vector 3 Transform E coli and analyze the transformants for the vector with 2 copies of your insert 4 Isolate and digest this vector with 2 copies of your gene with BamH I and Bgl II to release a cassette with 2 copies of your gene optional 5 Digest the vector with 2 copies of your gene with BamH I and ligate 1 or 2 copies see Step 4 of the expression cassette into the vector 6 Transform E coli and analyze the transformants for the vector with 3 or 4 copies of your insert 7 Repeat until the desired multimer is reached 25 Continued on next page Constructing n Vitro Multimers Continued Controls Important Digestion of Recombinant pPIC6 To evaluate your transformants and expression data later on we recommend transforming Pichia with pPIC6 the parent vector and pPIC6 containing one copy of your gene of interest This will allow you to compare expression levels to see if multiple copies significantly increase the amount of protein produced Also if you elect to determine how many copies of your gene are ina recombinan
27. dine tag R930 25 Anti His C term HRP requires the free carboxyl group for R931 25 AnticHis C term AP detection Lindner et al 1997 RIE HHHHHH COOH 34 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com SDS Certificate of Analysis Limited Warranty 35 Safety Data Sheets MSDSs are available on our website at www invitrogen com sds The Certificate of Analysis provides detailed quality control and product qu
28. e 19 for recipe Blasticidin antibiotic page 21 Low Salt LB plates containing 100 ug mL blasticidin see page 19 for recipe YPDS plates containing the appropriate concentration of blasticidin see page 20 for recipe 50 mL conical centrifuge tubes 15 mL polypropylene tubes TM ProBond Purification System optional see page 33 for ordering Product Overview Description of the System Experimental Overview Introduction pPIC6 A B and C are 3 4 kb vectors used to express recombinant proteins in Pichia pastoris The vector allows high level methanol inducible expression of the gene of interest in Pichia and can be used in any Pichia strain including the X 33 strain supplied with the kit pPIC6 contains the following elements e 5 fragment containing the AOX1 promoter for tightly regulated methanol induced expression of the gene of interest Ellis et al 1985 Koutz et al 1989 Tschopp et al 1987a e Blasticidin resistance gene Kimura et al 1994 Takeuchi et al 1958 Yamaguchi et al 1965 for selection in both E coli and Pichia e C terminal peptide containing the c myc epitope and a polyhistidine 6xHis tag for detection and purification of a recombinant fusion protein if desired e Three reading frames to facilitate in frame cloning with the C terminal peptide The control plasmid pPIC6 lacZ is included for use as a positive control for expression The following table describes the
29. entrifuge 2 minutes and remove the ethanol Centrifuge again for 1 minute remove residual ethanol and air dry the pellet Resuspend pellet in 4 uL sterile water Save on ice if you plan to ligate your insert immediately or you can store at 20 C Proceed to Ligating Multimers into Linearized Vector You may wish to combine the ligation reaction with the restriction enzyme digestion to enrich for head to tail multimers Use the reaction buffer for the restriction enzymes and add 1 mM ATP to the reaction in order to ensure ligase activity Perform the reaction at 37 C T4 ligase will retain most of its activity in the restriction buffer As head to head and tail to tail multimers form they will be digested increasing the likelihood of obtaining head to tail multimers over time You are now ready to ligate the mixture of multimers generated in Step 10 above into dephosphorylated linearized vector 1 Set up the following ligation reactions Dephosphorylated vector page 28 Step 10 4 uL Expression cassette multimers Step 10 above 4 uL 10X Ligation Buffer 1 uL T4 DNA Ligase 2 5 units uL 1 uL Total volume 10 uL Continued on next page Constructing n Vitro Multimers Continued Ligating Multimers into Linearized Vector Continued Transformation into E coli For the vector only control Dephosphorylated vector 4 uL Sterile water 4 uL 10X Ligation Buffer 1 uL T4 DNA Ligase 2 5 units uL 1 u
30. es cerevisiae Mol Cell Bio 11 3060 3069 Izumi M Miyazawa H Kamakura T Yamaguchi I Endo T and Hanaoka F 1991 Blasticidin S Resistance Gene bsr A Novel Selectable Marker for Mammalian Cells Exp Cell Res 197 229 233 Kimura M Takatsuki A and Yamaguchi I 1994 Blasticidin S Deaminase Gene from Aspergillus terreus BSD A New Drug Resistance Gene for Transfection of Mammalian Cells Biochim Biophys Acta 1219 653 659 Koutz P J Davis G R Stillman C Barringer K Cregg J M and Thill G 1989 Structural Comparison of the Pichia pastoris Alcohol Oxidase Genes Yeast 5 167 177 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nuc Acids Res 15 8125 8148 Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biol 115 887 903 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 Lindner P Bauer K Krebber A Nieba L Kremmer E Krebber C Honegger A Klinger B Mocikat R and Pluckthun A 1997 Specific Detection of His tagged Proteins With Recombinant Anti His Tag scFv Phosphatase or scFv Phage Fusions BioTechniques 22 140 149 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spri
31. g mL stock solution Note It is necessary to include blasticidin in the medium for selection of Pichia transformants only Blasticidin may be omitted from the medium when performing expression studies Store YPD slants or plates containing blasticidin at 4 C The shelf life is one to two weeks Continued on next page Recipes Continued YPDS Blasticidin Yeast Extract Peptone Dextrose Medium with Sorbitol 1 liter Agar Breaking Buffer 1 yeast extract 2 peptone 2 dextrose glucose 1 M sorbitol 2 agar the appropriate concentration of blasticidin 1 Dissolve 10 g yeast extract 182 2 g sorbitol 20 g of peptone in 900 mL of water Add 20 g of agar Autoclave for 20 minutes on liquid cycle Add 100 mL of 20 dextrose filter sterilize dextrose before use OS Cool solution to 60 C and add the appropriate amount of blasticidin from a 10 mg mL stock solution Note It is necessary to include blasticidin in the medium for selection of Pichia transformants only Blasticidin may be omitted from the medium when performing expression studies Store YPDS slants or plates containing blasticidin at 4 C The shelf life is one to two weeks 50 mM sodium phosphate pH 7 4 1 mM PMSF phenylmethylsulfonyl fluoride You may use other protease inhibitors 1mM EDTA 5 glycerol 1 Prepare a stock solution of your desired protease inhibitors and store appropriately Follow manufacturer s recommendations 2 For
32. iable Method to Create Tandem Arrays of Short DNA Sequences BioTechniques 13 780 789 Rudert W A and Trucco M 1990 DNA Polymers of Protein Binding Sequences Generated by Polymerase Chain Reaction Nucleic Acids Res 18 6460 Simpson R T Thoma F and Brubaker J M 1985 Chromatin Reconstituted from Tandemly repeated Cloned DNA Fragments and Core Histones A Model System for the Study of Higher order Structure Cell 42 799 808 Takeshita S Tezuka K i Takahashi M Honkawa H Matsuo A Matsuishi T and Hashimoto Gotoh T 1988 Tandem Gene Amplification in vitro for Rapid and Efficient Expression in Animal Cells Gene 71 9 18 Taylor W H and Hagerman P J 1987 A General Method for Cloning DNA Fragments in Multiple Copies Gene 53 139 144 32 Accessory Products Introduction The following products may be used with the pPIC6 vectors For details visit www invitrogen com or contact Technical Support see page 35 Item Amount Catalog no ProBond Purification System 6 purifications K850 01 50 mL R801 01 ProBond Resin 150 mL R801 15 ues see a 21 x 50 pL 3030 03 PureLink HiPure Plasmid Miniprep Kit 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 S N A P Gel Purification Kit 25 preps K1999 25 B Gal Assay Kit 80 mL K1455 01 B Gal Staining Kit 1 kit K1465 01 Blasticidin 50 mg R210 01 Positope Con
33. ine tag The vector is supplied in three reading frames to facilitate cloning Refer to the diagrams on pages 4 6 to develop a cloning strategy If you wish to express your protein WITHOUT the C terminal peptide be sure to include a stop codon pPIC6 A B and C contain unique Bgl ll and BamH I sites to allow construction of plasmids containing multiple copies of your gene For information on how to construct multimers refer to the Appendix pages 25 32 Continued on next page Cloning into pPIC6 A B and C Continued Multiple Cloning Site of pPIC6 A 811 871 931 991 1042 1098 1158 1218 Sac Il Not Apa Below is the multiple cloning site for pPIC6 A Restriction sites are labeled to indicate the cleavage site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of pPIC6 A is available for downloading from www invitrogen com or from Technical Support see page 35 For a map and a description of the features of pPIC6 refer to the Appendix pages 16 17 5 end of AOX1 MRNA 5 AOX7 priming site AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA 1 CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT Sfu EcoR Pml Sfi Asp718 Kpn I Xho I I I 1 ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTA
34. invitrogen pPIC6 A B and C Pichia expression vectors for selection on blasticidin and purification of recombinant proteins Catalog no V210 20 Rev date 7 June 2010 Manual part no 25 0344 MAN0000164 ii Table of Contents Kit Contents and Storag sree iE ier a n R EAEN EEEE Ee EEES OTE EE EES EEE ESEE EE iv Calda olo Ufo Lo y Pa 1 Product OVerVi EN Tona aces tances AE OAA ee a as ier lee OS ia taal 1 Method A A raaa naaran Arae aE Aaa E rira A haea NO NRE n Eisa EaR eRENAET 2 Cloning into pPIC6 A B and Eee sein opeet aea ESEE e Ear eae EE eaS EO RAS SREE ESE eE AAEE Eeee ISERE E ReiS SiE 2 Pichia Transtormation a a a eataeduadinininb att 8 EXPrESSIOO IE PICHIA iio cria E OEA e ib Ps o Casey tats 12 PU Nom a a e a A A ar rer rere reir eet ferret A rer Rererren ee freee AR ee Trey ARAA 14 PDD e aLr E ccuencvecevendwantwendudanventuanaudacwencvdndvieaveaddacweacaauedeaduanasdadwacsdaaduineutsaiuataiwe 16 PERICO Vector rai di tao ita des 16 RA AA A A A O NE 18 RECIPES nai ia EEEE 19 EC AAA es ele che ta A ARE aea aAA a Raa aAa aa O a E aia Ei 21 Lithium Chloride Transformation Method cssssessssesecceseeseeecseesececseesceecseeseeecseseeessesseeesnesseeeeneees 23 Constructing In Vitro MultimerS snoite iier nisieete iiaee Srta EEEo Ei ESAEREN eiA eraa RES iebes ine ESEE 25 ACCESSORY Prod Uta nni aeea teins esas ani Ua hase Ea EERE dd EE 33 Techni al Supportissts lt c senin eines steed A R oE AE eE AE EESE EEEE E S EE E E
35. l to tail and head to head orientation After digestion with these two restriction enzymes you will have a mixture of multimers containing 1 2 3 etc copies of your gene that can be ligated into BamH I linearized recombinant pPIC6 1 Ze Set up a 20 uL ligation reactions as follows Bgl Il BamH I digested expression cassette 15 pL Sterile water 2 uL 10X Ligation Buffer with ATP 2 pL T4 DNA Ligase 2 5 units uL 1 uL Incubate at 16 C for 2 5 hours Continued on next page 28 Constructing n Vitro Multimers Continued Ligation and Digestion of Expression Cassetie Continued Note Ligating Multimers into Linearized Vector 29 10 Heat inactivate the ligase by incubating at 65 C for 20 minutes Add the following reagents for restriction enzyme digestion cut back Note that BamH I and Bgl II may be used with the same reaction buffer Sterile water 23 uL 10X restriction enzyme buffer 5 uL Bgl II 10 units pL 1 uL BamH I 10 units uL 1 uL Incubate the reaction at 37 C for 2 hours Add 50 uL of phenol chloroform and extract the restriction enzyme digestion to remove the enzymes Transfer the aqueous solution to a new microcentrifuge tube To ethanol precipitate the DNA add 5 uL of 3 M sodium acetate and 110 uL of 100 ethanol Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4 C Carefully decant the supernatant Wash the nucleic acid pellet with 80 ethanol c
36. minutes at 4 C Resuspend the pellet with 500 mL of ice cold sterile water Centrifuge the cells as in Step 3 and resuspend the pellet with 250 mL of ice cold sterile water Centrifuge the cells as in Step 3 and resuspend the pellet in 20 mL of ice cold 1 M sorbitol Centrifuge the cells as in Step 3 and resuspend the pellet in 1 mL of ice cold 1 M sorbitol for a final volume of approximately 1 5 mL Keep the cells on ice and use that day Do not store cells 1 Mix 80 uL of the cells from Step 6 above with 5 10 ug of linearized pPIC6 DNA in 5 10 pL sterile water and transfer them to an ice cold 0 2 cm electroporation cuvette Incubate the cuvette with the cells on ice for 5 minutes Pulse the cells according to the parameters for yeast Saccharomyces cerevisiae as suggested by the manufacturer of the specific electroporation device being used Immediately add 1 mL of ice cold 1 M sorbitol to the cuvette Transfer the cuvette contents to a sterile 15 mL tube Let the tube incubate at 30 C without shaking for 1 to 2 hours Spread 50 200 uL each on separate labeled YPDS plates containing the appropriate concentration of blasticidin Incubate plates for 2 to 3 days at 30 C until colonies form Pick 10 20 colonies and purify streak for single colonies on fresh YPD or YPDS plates containing the appropriate concentration of blasticidin 10 Pichia Transformation Continued ee Nx D Y _f Va And Note
37. multimers in Pichia transformation into E coli Problem Possible Reason Solution No multimers or low number of CIAP defective Use fresh CIAP multimers in your vector after Add more CIAP Add 1 unit of CIAP and incubate 15 more minutes at 37 C This is somewhat risky as CIAP can degrade the ends of your DNA Not enough insert DNA Add more BamH I Bgl II expression to ligate cassette to your ligation Construct is unstablein Decrease the number of cassettes in E coli the vector Multimers are too long to Try ligating each expression cassette ligate efficiently stepwise Recombinant vector rearranges Construct is unstablein Decrease the number of cassettes in and deletions are detected E coli the vector No blasticidin resistant Pichia Integration efficiency is Transform using more DNA and or transformants low do multiple transformations with more DNA and cells For More There are a number references in the literature you can consult to optimize Information synthesis of in vitro multimers A partial list is provided below Cohen B and Carmichael G G 1986 A Method for Constructing Multiple Tandem Repeats of Specific DNA Fragments DNA 5 339 343 Eisenberg S Francesconi S C Civalier C and Walker S S 1990 Purification of DNA Binding Proteins by Site specific DNA Affinity Chromatography Methods Enzymol 182 521 529 Graham G J and Maio J J 1992 A Rapid and Rel
38. ng Harbor Laboratory Press Scorer C A Buckholz R G Clare J J and Romanos M A 1993 The Intracellular Production and Secretion of HIV 1 Envelope Protein in the Methylotrophic Yeast Pichia pastoris Gene 136 111 119 Takeuchi S Hirayama K Ueda K Sakai H and Yonehara H 1958 Blasticidin S A New Antibiotic The Journal of Antibiotics Series A 11 1 5 Tschopp J F Brust P F Cregg J M Stillman C and Gingeras T R 1987a Expression of the lacZ Gene from Two Methanol Regulated Promoters in Pichia pastoris Nucleic Acids Res 15 3859 3876 Continued on next page 38 References Continued Yamaguchi H Yamamoto C and Tanaka N 1965 Inhibition of Protein Synthesis by Blasticidin S I Studies with Cell free Systems from Bacterial and Mammalian Cells J Biochem Tokyo 57 667 677 Zaret K S and Sherman F 1984 Mutationally Altered 3 Ends of Yeast CYC1 mRNA Affect Transcript Stability and Translational Efficiency J Mol Biol 177 107 136 2009 Life Technologies Corporation All rights reserved 39
39. ns on ice Small scale purifications using the 2 mL ProBond columns and buffers can be done at room temperature on the bench top For large scale purifications all reagents must be kept at 4 C Express your protein using a small scale culture 10 20 mL for Mut strains 100 200 mL for Mut and the optimal conditions for expression if determined Once your protein is expressed follow the protocol below to prepare a cell lysate for TM chromatography on ProBond Prepare Breaking Buffer BB as described in the Recipes page 20 1 Wash cells once in BB by resuspending them and centrifuging 5 10 minutes at 3 000 x g at 4 C 2 Resuspend the cells to an ODs of 50 100 in BB Add an equal volume of acid washed glass beads 0 5 mm Estimate volume by displacement 4 Vortex the mixture for 30 seconds then incubate on ice for 30 seconds Repeat 7 more times Alternating vortexing with cooling keeps the cell extracts cold and reduces denaturation of your protein Centrifuge the sample at 4 C for 5 10 minutes at 12 000 x g Transfer the clear supernatant to a fresh container and analyze for your protein The total protein concentration should be around 2 3 mg mL 7 Save the pellet and extract with 6 M urea or 1 Triton X 100 to check for insoluble protein Continued on next page 14 Purification Continued Sample Application Native Conditions Sample Application Denaturing Conditions Note
40. of sterile water and centrifuge at 1 500 x g for 10 minutes at room temperature Resuspend the cell pellet in 1 mL of 100 mM LiCl and transfer the suspension to a 1 5 mL microcentrifuge tube Pellet the cells at maximum speed for 15 seconds and remove the LiCl with a pipet Resuspend the cells in 400 uL of 100 mM LiCl Dispense 50 uL of the cell suspension into a 1 5 mL microcentrifuge tube for each transformation and use immediately Do not store on ice or freeze at 20 C 23 Continued on next page Lithium Chloride Transformation Method Continued Transformation 1 MOE ORS ral FON 0 Boil a 1 mL sample of single stranded DNA for 5 minutes then quickly chill on ice Keep on ice Note It is not necessary to boil the carrier DNA prior to each use Store a small aliquot at 20 C and boil every 3 4 times the DNA is thawed Centrifuge the cells from Step 6 above and remove the LiCl with a pipette For each transformation add the following reagents IN THE FOLLOWING ORDER to the cells PEG shields the cells from the detrimental effects of the high LiCl concentration 50 PEG 240 uL 1M LiCl 36 pL 2 mg mL single stranded DNA 25 uL Plasmid DNA in 50 uL sterile water 5 10 ug Vortex each tube vigorously until the cell pellet is completely mixed 1 minute Incubate the tube at 30 C for 30 minutes without shaking Heat shock in a water bath at 42 C for 20 25 minutes Centrifuge the cells at 3 800 to 6 8
41. ollowing address Bennett Cohen Ph D Research Corporation Technologies 101 North Wilmot Road Suite 600 Tucson Arizona 85711 3335 Tel 520 748 4443 Fax 520 748 0025 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology New York Greene Publishing Associates and Wiley Interscience Ellis S B Brust P F Koutz P J Waters A F Harpold M M and Gingeras T R 1985 Isolation of Alcohol Oxidase and Two other Methanol Regulatable Genes from the Yeast Pichia pastoris Mol Cell Biol 5 1111 1121 Evans G L Lewis G K Ramsay G and Bishop V M 1985 Isolation of Monoclonal Antibodies Specific for c myc Proto oncogene Product Mol Cell Biol 5 3610 3616 Gietz R D and Schiestl R H 1996 Transformation of Lithium Treated Yeast Cells and the Selection of Auxotrophic and Dominant Markers In Methods in Molecular Biology 1 H Evans ed Totowa NJ Humana Press Henikoff S and Cohen E H 1984 Sequences Responsible for Transcription Termination on a Gene Segment in Saccharomyces cerevisiae Mol Cell Biol 4 1515 1520 Higgins D R and Cregg J M 1998 Pichia Protocols In Methods in Molecular Biology Vol 103 J M Walker ed Humana Press Totowa NJ Irniger S Egli C M and Braus G H 1991 Different Classes of Polyadenylation Sites in the Yeast Saccharomyc
42. on usage in Pichia is believed to be similar to Saccharomyces cerevisiae Many Saccharomyces genes have proven to be functional in Pichia The premature termination of transcripts because of AT rich regions has been observed in Pichia and other eukaryotic systems Henikoff and Cohen 1984 Irniger et al 1991 Scorer et al 1993 Zaret and Sherman 1984 If you have problems expressing your gene check for premature termination by northern analysis and check your sequence for AT rich regions It may be necessary to change the sequence in order to express your gene Scorer et al 1993 The native 5 end of the AOX1 mRNA is noted in the diagram for each multiple cloning site This information is needed to calculate the size of the expressed mRNA of the gene of interest if you need to analyze mRNA for any reason Your insert should contain a Kozak translation initiation sequence with an ATG start codon for proper initiation of translation Kozak 1987 Kozak 1991 Kozak 1990 An example of a Kozak consensus sequence is provided below Note that other sequences are possible but the G or A at position 3 and the G at position 4 are the most critical for function shown in bold The ATG initiation codon is shown underlined G A NNATGG pPIC6 is a terminal fusion vector To express your gene as a recombinant fusion protein you must clone your gene in frame with the C terminal peptide containing the c myc epitope and the polyhistid
43. pH of the aqueous solution should not exceed 7 to prevent inactivation of blasticidin e Do not subject stock solutions to freeze thaw cycles do not store in a frost free freezer e Upon thawing use what you need and discard the unused portion Continued on next page Blasticidin Continued Molecular Weight The formula for blasticidin is C 7H2 NgOs HCl and the molecular weight is Formula and 458 9 The diagram below shows the structure of blasticidin Structure NH2 ry A N HOOC O CH3 HCl SOA NH NH2 O 22 Lithium Chloride Transformation Method Introduction This is a modified version of the procedure described for S cerevisiae Gietz and Schiestl 1996 and is provided as an alternative to transformation by electroporation Transformation efficiency is between 10 to 10 cfu ug linearized DNA Preparing Lithium acetate does not work with Pichia pastoris Use only lithium chloride Solutions e 1MLIClin distilled deionized water Filter sterilize Dilute as needed with sterile water 50 polyethylene glycol PEG 3350 in distilled deionized water Filter sterilize Store in a tightly capped bottle 2 mg mL denatured sheared salmon sperm DNA in TE 10 mM Tris HCl pH 8 0 1 0 mM EDTA Store at 20 C Preparing Cells 1 Grow a 50 mL culture of Pichia pastoris in YPD at 30 C with shaking to an OD o of 0 8 to 1 0 approximately 10 cells mL Harvest the cells wash with 25 mL
44. proceed to Pichia Transformation next page Pichia Transformation Introduction Blasticidin Selection Method of Transformation Pichia EasyComp Transformation Kit Note Important You should now have your gene cloned into one of the pPIC6 vectors Your construct should contain a Kozak consensus sequence initiation ATG and be correctly fused to the C terminal peptide This section provides general guidelines to prepare plasmid DNA transform your Pichia strain and select for blasticidin resistant clones We generally use 300 pg mL blasticidin to select for transformants when using the X 33 Pichia strain If you wish to transform your pPIC6 construct into another Pichia strain note that selection conditions may vary We recommend performing a dose response curve to determine the appropriate concentration of blasticidin to use for selection of transformants in your strain We recommend electroporation for transformation of Pichia with pPIC6 A B or C Electroporation yields 10 to 10 transformants per ug of linearized DNA and does not destroy the cell wall of Pichia If you do not have access to an electroporation device use the LiCl protocol on page 23 or the Pichia TM EasyComp Transformation Kit available from Invitrogen see below If you wish to perform chemical transformation of your Pichia strain with pPIC6 A B or C the Pichia EasyComp Transformation Kit is available from Invitrogen see
45. processing including polyadenylation for increased mRNA stability TEF1 promoter GenBank accession numbers Transcription elongation factor 1 gene promoter from Saccharomyces cerevisiae that D12478 D01130 drives expression of the blasticidin resistance gene in Pichia EM7 promoter Synthetic prokaryotic promoter that drives constitutive expression of the blasticidin resistance gene in E coli Blasticidin resistance gene bsd Allows selection of transformants in E coli and Pichia CYC1 transcription termination region 3 end of the Saccharomyces cerevisiae CYC1 gene that allows efficient 37 mRNA processing of the blasticidin resistance gene for increased stability pUC origin Allows replication and maintenance of the plasmid in E coli pPIC6 lacZ Vector Description pPIC6 lacZ is a 6386 bp control vector containing the gene for B galactosidase The vector was constructed by ligating a 3 1 kb BstB I Not I fragment containing the lacZ gene into the pPIC6 B vector Map of pPIC6 lacZ The figure below summarizes the features of the pPIC6 lacZ vector The complete sequence for pPIC6 lacZ is available for downloading from www invitrogen com or from Technical Support see page 35 5 lacZ c myc epitope 6xHis Stop Comments for pPIC6 lacZ 6386 nucleotides 5 AOX1 promoter region bases 1 942 5 AOX7 priming site bases 855 875 LacZ ORF bases 941 3997 c myc epi
46. s are possible Note that for the enzymes listed below the cleavage site is the same for versions A B and C of pPIC6 Be sure that your insert does not contain the restriction site you wish to use to linearize your vector Enzyme Restriction Site bp Supplier Sac I 209 Many Pme I 414 New England Biolabs BstX I 707 Many Continued on next page Pichia Transformation Continued Restriction Digest Preparing Pichia for Electroporation Transformation by Electroporation 1 Ze Digest 5 10 ug of plasmid DNA with one of the enzymes listed above Check a small aliquot of your digest by agarose gel electrophoresis for complete linearization If the vector is completely linearized heat inactivate or add EDTA to stop the reaction phenol chloroform extract once and ethanol precipitate using 1 10 volume 3 M sodium acetate and 2 5 volumes of 100 ethanol Centrifuge the solution to pellet the DNA wash the pellet with 80 ethanol air dry and resuspend the DNA in 10 uL sterile deionized water Use immediately or store at 20 C Follow the procedure below to prepare your Pichia pastoris strain for electroporation 1 Grow 5 mL of your Pichia pastoris strain in YPD in a 50 mL conical tube at 30 C overnight Inoculate 500 mL of fresh medium in a 2 liter flask with 0 1 0 5 mL of the overnight culture Grow overnight again to an OD w 1 3 1 5 Centrifuge the cells at 1 500 x g for 5
47. ssettes for Multimerization 27 TM The S N A P Gel Purification Kit available from Invitrogen see page 33 allows you to rapidly purify DNA fragments from regular agarose gels Alternatively TM you may use glass milk To use the S N A P Gel Purification Kit follow the steps below 1 Electrophorese your digest from Step 1 above on a 1 to 5 regular TAE agarose gel Note Do not use TBE to prepare agarose gels Borate interferes with the sodium iodide step below Cut out the gel slice containing the PCR product and melt it at 65 C in 2 volumes of the 6 M sodium iodide solution Add 1 5 volumes Binding Buffer Load solution no more than 1 mL at a time from Step 3 onto a S N A P column Centrifuge 1 minute at 3000 x g in a microcentrifuge and discard the supernatant If you have solution remaining from Step 3 repeat Step 4 Add 900 uL of the Final Wash Buffer Centrifuge 1 minute at full speed in a microcentrifuge and discard the flow through Repeat Step 7 Elute the purified DNA in 15 uL of sterile water Store on ice if proceeding immediately to Ligating the Expression Cassette next page Store at 20 C for long term storage Continued on next page Constructing n Vitro Multimers Continued Dephosphorylating Dephosphorylation of the BamH I digested vector is necessary to prevent self ligation the Vector Ligating and Digesting the Expression Cassette 1 Dy LOT oR
48. sticidin Grow overnight at 37 C Isolate plasmid DNA and digest with Bgl Il and BamH I to release any multimers from pPIC6 Be sure to include Bgl II BamH I digested pPIC6 as a control It is possible to get vector rearrangements and deletions with large recombinant vectors in E coli Including Bgl II BamH I digested pPIC6 will allow you to detect these rearrangements deletions in the vector backbone Analyze your digests on a 1 agarose gel You should see bands corresponding to 1 copy 2 copies 3 copies etc of your expression cassette along with the vector backbone The number of copies you obtain may depend on how well a large vector is tolerated by the host strain Once you have identified plasmids with multiple copies of your expression cassette be sure to purify by streaking for single colonies and confirming your construct Prepare frozen glycerol stocks of E coli containing each of your multimeric constructs Prepare at least 100 ug of each plasmid for transformation into Pichia You need more DNA because you will be transforming with uncut plasmid DNA Transformation efficiency is about 1 to 2 orders of magnitude less for uncut versus linearized DNA Proceed to Pichia Transformation page 8 Use the outgrowth protocol on page 11 to isolate transformants Continued on next page Constructing n Vitro Multimers Continued Troubleshooting The table below will help you optimize formation and isolation of
49. t by dot or Southern blot the strain with the parent vector will control for background hybridization and the strain with the single copy gene will provide a signal to normalize your data Once you have created a pPIC6 plasmid containing multimers note that this plasmid cannot be linearized because any enzyme that cuts in the 5 AOX1 region will cut in all of the 5 AOXT regions present in the multimer You can transform with uncut plasmid but you will need to use 50 100 pg of DNA to compensate for the 10 to 100 fold drop in transformation efficiency However with selection on blasticidin any transformants you obtain will probably contain your construct For best results e Use electroporation to transform your cells e Use at least 50 ug plasmid DNA for each transformation e Plate out all of the transformation mix on several YPDS plates containing the appropriate concentration of blasticidin You will need to use the optional outgrowth procedure on page 11 Set up two separate digests of recombinant pPIC6 containing one copy of your gene 1 Double digest 1 2 ug of recombinant pPIC6 in 20 uL with 10 units each of Bgl II and BamH I Proceed to Producing Expression Cassettes for Multimerization Step 1 2 Digest 2 ug of recombinant pPIC6 in 20 uL with 10 units of BamH I only Proceed to Dephosphorylating the Vector Step 1 Continued on next page 26 Constructing n Vitro Multimers Continued Producing Expression Ca
50. tern blot analysis to detect your protein if expression is low or not enough protein was loaded onto the column Refer to the ProBond Purification System manual for a guide to troubleshoot chromatography You may find it necessary to scale up your purification to obtain sufficient amounts of purified protein Adjust the pH and NaCl concentration of your lysate with 1 10 volume of 10X Stock Solution B ProBond Purification Kit before adding it to the column The pH should be 2 7 5 and the NaCl concentration should be 500 mM Using 10X Stock Solution B to adjust the pH and the ionic strength keeps the total volume small for sample application Appendix pPIC6 Vector Map of pPIC6 The figure below summarizes the features of the pPIC6 A B and C vectors The vector sequences for pPIC6 A B and C are available for downloading from www invitrogen com or from Technical Support see page 35 See the next page for a description of the features of the vector EcoR Pml Sfi Asp718 32 c myc epitope 6xHis Stop Sac Il sE xXx H Comments for pPIC6 A 3382 nucleotides Bgl Il 5 AOX1 promoter region bases 1 942 5 AOX1 priming site bases 855 875 The restriction site between Multiple cloning site bases 932 1011 Not and the c myc epitope is c myc epitope bases 1012 1041 different in each version of Polyhistidine 6xHis tag bases 1057 1074 pPIC6 3 AOX7 priming site bases 1160 1180 Apa
51. tes using the recipe in the Appendix page 19 Failure to lower the salt content of your LB medium will result in non selection due to inhibition of the drug We recommend that you sequence your construct to confirm that your gene is in the correct orientation for expression cloned in frame with the C terminal peptide and contains an ATG start codon and a stop codon We suggest using the 5 AOX1 Pichia and 3 AOX1 Pichia primer sequences Refer to the diagrams on pages 4 6 for the sequences and location of the priming sites Once you have identified the correct clone be sure to purify the colony and make a glycerol stock for long term storage It is also a good idea to keep a DNA stock of your plasmid at 20 C 1 Streak the original colony on Low Salt LB plate containing 100 ng mL blasticidin Incubate the plate at 37 C overnight 2 Isolate a single colony and inoculate into 1 2 mL of Low Salt LB containing 100 pg mL blasticidin Grow the culture to mid log phase ODs 0 5 0 7 Mix 0 85 mL of culture with 0 15 mL of sterile glycerol and transfer to a cryovial 5 Store at 80 C Once you have cloned and sequenced your insert generate enough plasmid DNA to transform Pichia 5 10 ug of each plasmid per transformation We recommend isolating plasmid DNA using the PureLink HiPure Plasmid Miniprep Kit or the PureLink HiPure Plasmid Midiprep Kit or equivalent see page 33 Once you have purified plasmid DNA
52. tope bases 4016 4045 Polyhistidine 6xHis tag bases 4061 4078 3 AOX7 priming site bases 4164 4184 AOX7 transcription termination region bases 4082 4423 TEF1 promoter bases 4424 4832 EM7 promoter bases 4837 4903 Blasticidin resistance gene bases 4904 5302 CYC1 transcription termination region bases 5331 5648 pUC origin bases 5659 6332 complementary strand 18 Recipes Low Salt LB Medium with Blasticidin YPD Blasticidin 19 10 g Tryptone 5 g NaCl 5 g Yeast Extract 1 Combine the dry reagents above and add deionized distilled water to 950 mL Adjust pH to 7 0 with IN NaOH Bring the volume up to 1 liter For plates add 15 g L agar before autoclaving Autoclave on liquid cycle at 15 psi and 121 C for 20 minutes Allow the medium to cool to at least 55 C before adding the blasticidin to 100 pg mL final concentration Store plates at 4 C in the dark Plates containing blasticidin are stable for up to 2 weeks Yeast Extract Peptone Dextrose Medium 1 liter 1 yeast extract 2 peptone 2 dextrose glucose 2 agar the appropriate concentration of blasticidin 1 Dissolve 10 g yeast extract 20 g of peptone in 900 mL of water 2 Include 20 g of agar if making YPD slants or plates 3 Autoclave for 20 minutes on liquid cycle 4 Add 100 mL of 20 dextrose filter sterilize dextrose before use 5 Cool solution to 60 C and add the appropriate amount of blasticidin from a 10 m
53. trol Protein 5 ug R900 50 Anti Mouse WB7103 WesternBreeze Chromogenic Kit Anti Rabbit WB7105 Anti Goat WB7107 Anti Mouse WB7104 WesternBreeze Chemiluminescent Kit Anti Rabbit WB7106 Anti Goat WB7108 Countess Automated Cell Counter 1 each C10227 Continued on next page 33 Accessory Products Continued Other Pichia Other Pichia products available from Invitrogen are described below Products Item Amount Catalog no X 33 Pichia strain 1 stab C180 00 KM71H Pichia strain 1 stab C182 00 SMD1168H Pichia strain 1 stab C184 00 pPIC6a A B and C 20 ug each V215 20 pPICZ A B and C 20 ug each V190 20 pPICZa A B and C 20 ug each V195 20 Original Pichia Expression Kit 1 kit K1710 01 EasySelect Pichia Expression Kit 1 kit K1740 01 Pichia EasyComp Transformation Kit 1 kit K1730 01 Pichia Protocols 1 book G100 01 Antibodies If you do not have an antibody specific to your protein Invitrogen offers the Anti myc or Anti His C term antibodies to detect your recombinant fusion protein Horseradish peroxidase HRP and alkaline phosphatase AP conjugated antibodies are available for convenient one step detection Antibody Epitope Catalog no Anti myc Detects a 10 amino acid epitope derived R950 25 Anti myc HRP from c myc Evan et al 1985 R951 25 Anti myc AP POSE RIDE R952 25 Anti His C term Detects the C terminal polyhisti
54. u in furtherance of and reliance on such license You may not use the Expression Vectors for any commercial purpose without a license for such purpose from Research Corporation Technologies Inc Tucson Arizona Continued on next page 36 Purchaser Notification Continued Limited Use Label License No 74 Pichia pastoris Expression System Continued 37 Commercial purposes include any use of Expression Products or Expression Vectors in a Commercial Product any use of Expression Products or Expression Vectors in the manufacture of a Commercial Product any sale of Expression Products any use of Expression Products or the Expression Kit to facilitate or advance research or development directed to a Commercial Product and any use of Expression Products or the Expression Kit to facilitate or advance any research or development program the results of which will be directly applied to the development or manufacture of a Commercial Product Expression Products means products expressed with the Expression Kit or with the use of any Pichia expression vectors including the Expression Vector or host strains Commercial Product means any product intended for sale or commercial use Commercial entities may conduct their evaluation for one year at which time this license automatically terminates Commercial entities will be contacted by Research Corporation Technologies during the evaluation period regarding their desire for
Download Pdf Manuals
Related Search