APPENDIX 1 - PFCA SEQUENCES

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APPENDIX 1 - PFCA SEQUENCES

NATIVE PFCA SEQUENCE, ENTIRE OPEN READING FRAME

atgcttgaaatgatagataaatataatacccattttgtacaaacaaccaaaccttattac M L E M I D K Y N T H F V Q T T K P Y Y gaatttaatgtaactaatctaactaattccaaaaaaaaaaaaaaaaaaaaaaaaagggaa E F N V T N L T N S K K K K K K K K R E aatcacctgatcggttcaggtgaaaatatgcaaaaaaaggatgaaaaaaatataaaggat N H L I G S G E N M Q K K D E K N I K D tttcatataaatgattatgaaatagatgggaaaacaattcataataaagagaacaaggat F H I N D Y E I D G K T I H N K E N K D tcctttaaaatgaataaaaataaattaaacgataacgaagaattattttatatggacaat S F K M N K N K L N D N E E L F Y M D N atattatcttataaaccaaataaaaagaagttgtttacttattccttttccgaaaatgaa I L S Y K P N K K K L F T Y S F S E N E ggaaattctgaaaaagaagaaaccctttataattttaaaaatatgaaaaatataaatagc G N S E K E E T L Y N F K N M K N I N S gtacaaaataatattaacaagacctttttatataacaaattgaaaaatgtagattattat V Q N N I N K T F L Y N K L K N V D Y Y gagcatggttataattgggatataggtcaatgtaaaacagggaaatatcaatctcctgtt E H G Y N W D I G Q C K T G K Y Q S P V gatttacctatgaaagatttaaaggagagagaattaaaaaatataagtgatgtgtattta D L P M K D L K E R E L K N I S D V Y L aatttatttgacgatgacaattatgcatggaacaattataacaaaccatggatgaaagga N L F D D D N Y A W N N Y N K P W M K G gattttttttattattatgaatattttataaaaaaaattgttattaatagacaaaataat D F F Y Y Y E Y F I K K I V I N R Q N N atatttcaaataaaagctgcaagagatggaataataccatttggtgtgttatttactact I F Q I K A A R D G I I P F G V L F T T gaacaacctgctatgttttatgcagatcaaatccattttcatgctcctagcgaacataca E Q P A M F Y A D Q I H F H A P S E H T tttcaaggttcaggtaatagaagagaaattgaaatgcaaatatttcatagtacaaattat F Q G S G N R R E I E M Q I F H S T N Y ttttatgatatacaagatgataaatccaaatataaaaaaaaatacgggttacatatatat F Y D I Q D D K S K Y K K K Y G L H I Y aataatttaaaaaaaaattcaaaagaaacttcaaaaaaagattcaagtagatatcattct N N L K K N S K E T S K K D S S R Y H S tatcttatgtcctttctaatgaatagcttatcaaatgaacaattacaaaacaaatataat Y L M S F L M N S L S N E Q L Q N K Y N aaaaaaaaaagaataaaaaagatgaaaaaccaatatgaagtaatatctattacatttact K K K R I K K M K N Q Y E V I S I T F T agtgcagaaattaatgcttcaactattaatgcttttaagaaattaccatcagaaaaattt S A E I N A S T I N A F K K L P S E K F ctaagaactataataaatgtatcaagtgcagttcacgtcggctcaggtaataaataa L R T I I N V S S A V H V G S G N K -

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CODON OPTIMIZED PFCA1 (ADJUSTED FOR GLYCOSYLATION)

atgaaggatttgaaggaaagagaacttaagcaaattagtgatgtgtacttgaacctgttc M K D L K E R E L K Q I S D V Y L N L F gatgacgataactacgcatggaataactacaataagccttggatgaagggagatttcttc D D D N Y A W N N Y N K P W M K G D F F tactactacgaatactttattaagaaaatagttatcaacagacagaataacatctttcaa Y Y Y E Y F I K K I V I N R Q N N I F Q attaaggcagcaagggatggaattataccatttggtgttctgtttactacagaacaacct I K A A R D G I I P F G V L F T T E Q P gcaatgttttacgctgaccagatacacttccatgccccatcagaacacacattccaagga A M F Y A D Q I H F H A P S E H T F Q G tctggaaaccgtcgtgaaattgagatgcaaatctttcactctacaaattatttttatgac S G N R R E I E M Q I F H S T N Y F Y D attcaagacgacaaatccaagtataagaagaaatacggattgcacatatataacaacttg I Q D D K S K Y K K K Y G L H I Y N N L aagaagaactctaaagaaacaagcaagaaggattcttctaggtaccattcctacttaatg K K N S K E T S K K D S S R Y H S Y L M tcctttttgatgaactcactttccaacgagcaacttcaaaacaagtacaataagaagaaa S F L M N S L S N E Q L Q N K Y N K K K aggattaaaaagatgaaaaatcaatatgaagtgattagcatcacctttacatccgctgag R I K K M K N Q Y E V I S I T F T S A E atacaagcctctactattaatgcttttaagaagttgccttctgaaaagttcttgagaact I Q A S T I N A F K K L P S E K F L R T attattcaagtaagttccgctgttcacgtaggatcaggtaataag I I Q V S S A V H V G S G N K

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CODON OPTIMIZED PFCA2 (ADJUSTED FOR GLYCOSYLATION)

atgaagaatattaattcagtccaaaacaacatccaaaagacatttttatataacaagctt M K N I N S V Q N N I Q K T F L Y N K L aagaatgttgactattacgaacacggttataattgggatatcggacagtgtaaaaccggt K N V D Y Y E H G Y N W D I G Q C K T G aaatatcagtccccagttgatctgccaatgaaggatctgaaagaaagagagttgaagcag K Y Q S P V D L P M K D L K E R E L K Q atctctgatgtttacttgaatctgtttgacgatgataactatgcttggaacaactacaac I S D V Y L N L F D D D N Y A W N N Y N aaaccttggatgaaaggtgactttttctattactacgaatattttatcaaaaagattgtc K P W M K G D F F Y Y Y E Y F I K K I V atcaatagacaaaacaacatttttcaaattaaggccgctagagatggtattattcctttt I N R Q N N I F Q I K A A R D G I I P F ggtgttctgttcaccactgaacagccagcaatgttttatgcagaccaaattcactttcat G V L F T T E Q P A M F Y A D Q I H F H gctcctagtgagcacacttttcaaggttccggtaatagaagagaaattgagatgcaaatc A P S E H T F Q G S G N R R E I E M Q I tttcattctacaaactatttctacgatattcaagatgataaatccaagtataagaagaaa F H S T N Y F Y D I Q D D K S K Y K K K tatggtttgcatatctacaataacttgaagaagaactcaaaagaaacatcaaagaaagat Y G L H I Y N N L K K N S K E T S K K D tcctcaagataccattcctacttaatgtcatttctgatgaatagtttgtctaacgaacaa S S R Y H S Y L M S F L M N S L S N E Q ttgcaaaataagtacaataagaaaaagagaatcaaaaagatgaagaatcaatacgaggtt L Q N K Y N K K K R I K K M K N Q Y E V atctctattacttttacctctgccgaaattcaagcttccaccatcaatgctttcaaaaag I S I T F T S A E I Q A S T I N A F K K cttccatccgaaaagttcctgagaacaatcatccaggtcagttctgccgtccacgttgga L P S E K F L R T I I Q V S S A V H V G tctggaaataag S G N K

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CODON OPTIMIZED PFCA3 (ADJUSTED FOR GLYCOSYLATION)

atgttggaaatgattgataaatataatactcattttgttcagactacaaaaccatactac M L E M I D K Y N T H F V Q T T K P Y Y gagtttcaagtcactcaattgactaacagtaaaaagaaaaagaagaagaaaaagagagaa E F Q V T Q L T N S K K K K K K K K R E aaccatttgatcggttctggtgaaaacatgcagaaaaaggatgagaagaacattaaggat N H L I G S G E N M Q K K D E K N I K D tttcacattaacgattacgagatcgatggaaagactattcacaataaggaaaacaaggat F H I N D Y E I D G K T I H N K E N K D tccttcaagatgaataagaacaaattgaatgataacgaagaattgttttatatggataat S F K M N K N K L N D N E E L F Y M D N attttgagttacaagcctaataagaaaaagttgtttacttattctttctcagagaacgaa I L S Y K P N K K K L F T Y S F S E N E ggtaactcagagaaagaagaaacattgtataatttcaaaaatatgaagaacattaatagt G N S E K E E T L Y N F K N M K N I N S gttcagaacaacattcaaaagacctttttgtacaacaaattgaagaacgttgactactat V Q N N I Q K T F L Y N K L K N V D Y Y gagcacggttataattgggacatcggtcaatgtaagactggtaaataccaatccccagtt E H G Y N W D I G Q C K T G K Y Q S P V gatttgccaatgaaggacttgaaggaaagagagttgaaacaaatttctgatgtttatttg D L P M K D L K E R E L K Q I S D V Y L aatttgtttgatgacgataactacgcttggaacaactacaacaagccatggatgaagggt N L F D D D N Y A W N N Y N K P W M K G gactttttctactactatgagtattttattaaaaagattgtcattaacagacagaataac D F F Y Y Y E Y F I K K I V I N R Q N N attttccaaattaaagctgccagagatggtatcatcccatttggagttttgtttacaact I F Q I K A A R D G I I P F G V L F T T gaacaaccagcaatgttttacgctgatcagattcatttccatgcaccttcagaacataca E Q P A M F Y A D Q I H F H A P S E H T ttccagggtagtggaaatagaagagagattgaaatgcagatcttccatagtacaaactat F Q G S G N R R E I E M Q I F H S T N Y ttttatgacatccaagatgataagtccaaatataaaaagaagtacggattgcatatctat F Y D I Q D D K S K Y K K K Y G L H I Y aacaacttgaagaaaaattctaaggaaacttccaaaaaggattcttctagatatcactca N N L K K N S K E T S K K D S S R Y H S tacttgatgtccttcttgatgaactcattgtccaacgagcagttgcaaaacaagtacaac Y L M S F L M N S L S N E Q L Q N K Y N aagaagaagagaattaagaagatgaagaatcaatatgaagttatttccatcacttttaca K K K R I K K M K N Q Y E V I S I T F T tccgctgaaattcaggcttctacaattaatgcttttaagaagttgccatctgagaaattc S A E I Q A S T I N A F K K L P S E K F ttgagaactatcatccaagtctcttccgccgtccacgtcggatccggtaataag L R T I I Q V S S A V H V G S G N K

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APPENDIX 2 - CLUSTAL ALIGNMENT HCAII, MMCAV, PFCA*,

NGCA*

Conserved regions are marked in bold text.

HCAII/1-260 --- MmCAV/1-237 --- NgCA/1-252 --- PfCA/1-418 MLEMIDKYNTHFVQTTKPYYEFNVTNLTNSKKKKKKKKRENHLIGSGENMQKKDEKNIKD HCAII/1-260 --- MmCAV/1-237 --- NgCA/1-252 ---MPRFPRTLPRLTAVLLLACTAFS PfCA/1-418 FHINDYEIDGKTIHNKENKDSFKMNKNKLNDNEELFYMDNILSYKPNKKKLFTYSFSENE HCAII/1-260 ---MSHHWGYGKHNGPEHW---HKDFP-IAKGER MmCAV/1-237 ---GTR NgCA/1-252 AAAHGNHT---HWGYTGHDSPESWGNLSEEFRLCSTGKN PfCA/1-418 GNSEKEETLYNFKNMKNINSVQNNINKTFLYNKLKNVDYYEHGYN---WDIGQ-CKTGKY

HCAII/1-260 QSPVDIQSPVDIQSPVDIQSPVDIDTHTAKYDPSLKPLSVSY---DQ-ATSLRIL MmCAV/1-237 QSPINIQSPINIQSPINIQSPINIQWKDSVYDPQLAPLRVSY---DA-ASCRYLW NgCA/1-252 QSPVNIQSPVNIQSPVNIQSPVNITETV---SGKLPAIKVNY---K-PSMVDVE PfCA/1-418 QSPVDLQSPVDLQSPVDLQSPVDLPMKDLK-ERELKNISDVYLNLFDDDNYAWNNYNKPWMKGDFFYYYEYFIKKIVI

HCAII/1-260 NNGHAFNVEF---DDSQDKAVLKGGPLDGTYRLIQFHFHQFHFHQFHFHQFHFHWGSLDGQGSEHSEHSEHTVDK--KKYA SEH MmCAV/1-237 NTGYFFQVEF---DDSCEDSGISGGPLGNHYRLKQFHFHQFHFHQFHFHQFHFHWGATDEWGSEHSEHSEHAVDG--HTYP SEH NgCA/1-252 NNGHTIQVNY---PEGGNTLTVNG----RTYTLKQFHFHQFHFHQFHFHQFHFH---VPSENSENSENQIKG--RTFP SEN PfCA/1-418 NRQNNIFQIKAARDGIIPFGVLFTTEQPAMFYADQIHFHQIHFHQIHFHQIHFH---APSEHSEHSEHTFQGSGNRRE SEH

HCAII/1-260 AELHLVHELHLVHELHLVHWNT-KYGDFGKAVQQPDGLAVLGIFLKVGSAKPGLQKVVDVLDSIKTKGKSAD ELHLVH MmCAV/1-237 AELHLVHELHLVHELHLVHWNSTKYENYKKASVGENGLAVIGVFLKLGAHHQALQKLVDVLPEVRHKDTQVA ELHLVH NgCA/1-252 MEAHFVHEAHFVHEAHFVHLDEN---KQP---LVLAVLYEAGKTNGRLSSIWNVMPM--TAGKVKL EAHFVH PfCA/1-418 IEMQIFHEMQIFHEMQIFHSTNY---FYDIQDDKSKYKKKYGLHIYNNLKKNSKE EMQIFH

HCAII/1-260 FTNFDPRGLLPESLDYWTYPGSLTTPPLLECVTWIVLKEPISVSSEQVLKFRKLNFNGEG MmCAV/1-237 MGPFDPSCLMPACRDYWTYPGSLTTPPLAESVTWIVQKTPVEVSPSQLSMFRTLLFSGRG NgCA/1-252 NQPFDASTLLPKRLKYYRFAGSLTTPPCTEGVSWLVLKTYDHIDQAQAEKFTRAVGS--- PfCA/1-418 TS---KKDSSRYHSYLMSFLMNSLSNEQLQNKYNKKKRIKKMKNQYEVISI-TFTS HCAII/1-260 EPEELMVDNWRPAQPLKNRQIKA---SFK--- MmCAV/1-237 EEEDVMVNNYRPLQPLRDRKLRS---SFR--- NgCA/1-252 ---ENNRPVQPLNARVVIE--- PfCA/1-418 AEINASTINAFKKLPSEKFLRTIINVSSAVHVGSGNK

* HCAII – Homo sapiens CAII, MmCAV – Mus musculus CAV, PfCA – Plasmodium

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APPENDIX 3 – MEDIA AND STOCK SOLUTIONS

MEDIA RECIPES

YPD(Z) - YEAST EXTRACT PEPTONE DEXTROSE MEDIUM (+ZEOCIN)

(1 liter) 1% yeast extract 2% peptone 2% dextrose (glucose) + 2% agar + 100 μg/ml zeocin

1. Dissolve 10 g yeast extract and 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. Cool solution to ~60°C and add 100 ml of 10X D. Add 1.0 ml of 100 mg/ml zeocin, if desired. Liquid medium without zeocin can be stored at room temperature. Medium containing zeocin should be stored at +4°C in the dark. YPD slants or plates are stored at +4°C.The shelf life of medium is several months. Medium containing zeocin has a shelf life of one to two weeks.

YPDS(Z)AGAR - YEAST EXTRACT PEPTONE DEXTROSE MEDIUM

(1 liter) 1% yeast extract 2% peptone 2% dextrose (glucose) 1 M sorbitol 2% agar 100 μg/ml zeocin

1. Dissolve 10 g yeast extract, 20 g of peptone and 182.2 g sorbitol in 900 ml of water. 2. Add 20 g of agar.

3. Autoclave for 20 minutes on liquid cycle. 4. Add 100 ml of 10X D

5. Cool solution to ~60°C and add 1.0 ml of 100 mg/ml zeocin.

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BMGY AND BMMY - BUFFERED GLYCEROL-COMPLEX MEDIUM AND BUFFERED METHANOL-COMPLEX MEDIUM

(1 liter) 1% yeast extract 2% peptone 100 mM potassium phosphate, pH 6.0 1.34% YNB 4 x 10-5% biotin 1% glycerol or 0.5% methanol

1. Dissolve 10 g of yeast extract and 20 g of peptone in 700 ml water. 2. Autoclave 20 minutes on liquid cycle.

3. Cool to room temperature, then add the following and mix well: 100 ml 1 M potassium phosphate buffer, pH 6.0

100 ml 10X YNB 2 ml 500X B 100 ml 10X GY

4. For BMMY, add 100 ml 10X M instead of glycerol.

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STOCK SOLUTIONS

500X B (0.02% BIOTIN)

Dissolve 20 mg biotin in 100 ml of water.

Filter sterilize and store at +4°C. The shelf life of this solution is approximately one year.

10X D (20% DEXTROSE)

Dissolve 200 g of D-glucose in 1000 ml of water.

Autoclave for 15 minutes, or filter sterilize. The shelf life of this solution is approximately one year.

10X M (5% METHANOL)

Mix 5 ml of methanol with 95 ml of water.

Filter sterilize and store at +4°C. The shelf life of this solution is approximately two months.

10X GY (10% GLYCEROL)

Mix 100 ml of glycerol with 900 ml of water.

Sterilize either by filtering or autoclaving. Store at room temperature. The shelf life of this solution is greater than one year.

1 M POTASSIUM PHOSPHATE BUFFER, PH 6.0

Combine 132 ml of 1 M K2HPO4, 868 ml of 1 M KH2PO4 and confirm that the pH = 6.0 ± 0.1 (if the pH needs to be adjusted, use phosphoric acid or KOH)

Sterilize by autoclaving and store at room temperature. The shelf life of this solution is greater than one year.

10X YNB - YEAST NITROGEN BASE (WITHOUT AMINO ACIDS)

100g (NH4)2SO4 20g KH2PO4 10g MgSO4 2g NaCl 2g CaCl2 40mg inositol

Dissolve all ingredients in 1000ml water, and warm if necessary to solubilize. Filter sterilize, and store at +4ºC.

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APPENDIX 4 - ELECTROPORATION OF PICHIA

PREPARATION OF PICHIA FOR ELECTROPORATION

1. Grow 5 ml of your Pichia pastoris strain in YPD in a 50 ml conical at 30°C overnight.

2. 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 OD600 = 1.3-1.5.

3. Centrifuge the cells at 1500 x g for 5 minutes at +4°C. Resuspend the pellet with 500 ml of

ice-cold (0°C), sterile water.

4. Centrifuge the cells as in Step 3, then resuspend the pellet with 250 ml of ice-cold (0°C), sterile

water.

5. Centrifuge the cells as in Step 3, then resuspend the pellet in 20 ml of ice-cold (0°C) 1 M sorbitol.

6. Centrifuge the cells as in Step 3, then resuspend the pellet in 1 ml of ice-cold (0°C) 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.

TRANSFORMATION BY ELECTROPORATION

Set up two transformations per construct, if possible. (Requires more YPDS-plates)

Use “No DNA control”, cells electroporated without addition of DNA. Plate like the other tubes.

1. Mix 80 µl of the cells from Step 6 (above) with 5-10 µg of linearized DNA (in 5-10 µl sterile

water) and transfer them to an ice-cold (0°C) 0.2 cm electroporation cuvette.

2. Incubate the cuvette with the cells on ice for 5 minutes.

3. Pulse the cells according to the manufacturer's instructions for yeast.

4. Immediately add 1 ml of ice-cold 1 M sorbitol to the cuvette. Transfer the cuvette contents to a

sterile 15 ml tube.

5. Let the tube incubate at 30°C without shaking for 2 hours.

6. Add 1 ml YPD medium to each tube. Shake (~200 rpm) the cultures at 30°C.

7. After 1 hour, take one of the tubes and plate out the cells by spreading 200 µl on separate, labeled

150 mm YPDS plates containing 100 µg/ml Zeocin.

8. 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 µl on 150 mm YPDS plates containing 100 µg/ml Zeocin.

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APPENDIX 5 - FASTDNA SPIN KIT FOR SOIL PROTOCOL

SAMPLE PROCESSING

1. Centrifuge a 1.5 ml culture sample at 8,500 g and discard the supernatant. Resuspend the pellet in 978 µl Sodium Phosphate Buffer and transfer to the Lysing Matrix E Tube. Add 122 µl MT Buffer.

LYSIS

2. Secure tubes in FastPrep® Instrument and process for 3x30 seconds at speed 5.5. Cool the tubes on ice between processing for 3 minutes.

3. Centrifuge Lysing Matrix E Tubes at 14,000 g for 2x5 minutes. Turn the tubes 180º between the centrifugations.

4. Transfer supernatant to a clean tube. Add 250 µl PPS reagent and mix by shaking the tube by hand 10 times.

5. Centrifuge at 14,000 g for 5 minutes to pellet precipitate. Transfer supernatant to a clean 15ml tube. (Resuspend Binding Matrix Suspension before use.) Add 1ml Binding Matrix Suspension to the supernatant.

6. Place on a rotator or invert by hand for 2 minutes to allow binding of DNA to matrix. Place tube in a rack for 3 minutes to allow settling of silica matrix.

7. Remove 610 µl of supernatant being careful to avoid settled Binding Matrix. Discard supernatant. Resuspend Binding Matrix in the remaining amount of supernatant. Transfer approximately 650 µl of the mixture to a SPINTM Filter and centrifuge at 12,000 g for 5 minutes. Empty the catch tube and add the remaining supernatant to SPINTM Filter and spin again at 12,000 g for 5 minutes. Empty the catch tube.

8. Add 500µl SEWS-M to the SPIN Filter and centrifuge at 12,000 g for 2 minutes. Decant flow-through and replace SPINTM Filter in Catch tube. Centrifuge at 14,000 xg for 5 minutes to “dry” the matrix of residual SEWS-M wash solution

9. Remove SPINTM Filter and place in 1.5 µl eppendorf tube. Air dry the SPIN Filter for 5 minutes at room temperature.

10. Add 50µl DES (DNase/Pyrogen Free Water) and gently stir matrix on filter membrane with a pipette tip or vortex/finger flip to resuspend the silica for efficient elution of the DNA. Centrifuge at 14,000 xg for 1 minute to transfer eluted DNA to Catch Tube. DNA is now application-ready.

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APPENDIX 6 - IMMUNOBLOTTING

IMMUNOBLOTTING PROTOCOL

Prepare an SDS polyacrylamide gel designed to resolve your recombinant protein. Prepare your samples for electrophoresis.

1. Load your samples and electrophorese your SDS polyacrylamide gel.

2. Transfer proteins to nitrocellulose membrane electrophoretically. Use 20 mM Tris, 150 mM glycine, 20% v/v methanol as a transfer buffer. Transfer overnight at 10V with cooling.

3. Remove the nitrocellulose membrane and incubate it in 10 ml blocking buffer. Gently agitate using a rocker platform for 3 hours at room temperature.

4. Wash the nitrocellulose membrane in 20 ml PBST for 2 x 5 minutes with gentle agitation. 5. Transfer membrane to a tray containing the Anti-myc-HRP antibody diluted 1:5000 in 10 ml blocking buffer (2 µl of antibody diluted into 10 ml blocking buffer). Incubate with gentle agitation overnight.

6. Transfer membrane to a tray containing 20 ml PBST and wash for 2 x 5 minutes with gentle agitation.

7. Incubate the membranes with a HRP chromogen substrate (0.1 M citrate-phosphate buffer pH 5.0, containing o.o3% hydrogen peroxide and 1 mg/l para-phenylenediamine) for about 5 minutes.

DOT BLOT PROTOCOL

This protocol is used to quickly detect the presence of recombinant protein. Be sure to spot equivalent amounts of protein for each sample.

1. Use a dot blot apparatus to spot the samples onto nitrocellulose paper. 3. Allow membrane to air-dry.

4. Proceed with Immunoblotting Protocol, steps 4-8.

PBS - PHOSPHATE-BUFFERED SALINE

137 mM NaCl 2.7 mM KCl 10 mM Na2HPO4 1.8 mM KH2PO4

PBST - PHOSPHATE-BUFFERED SALINE + TWEEN 20

137 mM NaCl 2.7 mM KCl 10 mM Na2HPO4 1.8 mM KH2PO4 0.05% Tween-20, v/v

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APPENDIX 7 - BATCH PURIFICATION USING NI-NTA SUPERFLOW

1. Assemble the column according to the manufacturer’s instructions.

2. Completely resuspend a 50% Ni-NTA Superflow slurry and transfer 2 µl slurry to the column. 3. Allow the resin to settle. Do not allow resin to dry. If this should occur, resuspend resin in lysis buffer and repack the column.

4. Equilibrate column with 6 column volumes of lysis buffer.

5. Transfer the slurry to 50 ml sample and mix gently by shaking (100 rpm) at room temperature for 60 min.

6. Load the sample–Ni-NTA mixture into the column and collect the column flow-through. 7. Wash with 5 column volumes lysis buffer; collect wash fractions for SDS-PAGE analysis. 8. Wash with 5 column volumes wash buffer; collect wash fractions for SDS-PAGE analysis. 9. Elute the protein with 12 column volumes elution buffer. Collect the eluate in fractions of 1 ml size and analyze by SDS-PAGE.

BUFFERS FOR PURIFICATION UNDER NATIVE CONDITIONS

LYSIS BUFFER (1 LITER)

50 mM NaH2PO4 (6.90 g NaH2PO4·H2O (MW 137.99 g/mol)) 300 mM NaCl (17.54 g NaCl (MW 58.44 g/mol))

10 mM imidazole (0.68 g imidazole (MW 68.08 g/mol)) Adjust pH to 8.0 using NaOH.

WASH BUFFER (1 LITER)

ELUTION BUFFER (1 LITER)

APPENDIX 1 - PFCA SEQUENCES