* 1. Based on the chromatograms shown below, select columns that could be used to generate similar chromatograms.

  Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7
DEAE Sepharose
CM Sepharose
Ni Agarose
Phenyl Sepharose
MEP HyperCel
Superdex 200
SP Sepharose
Q FF
Butyl Sepharose

These are common protein purification chromatograms

These are common protein purification chromatograms

* 2. We would like to express and purify the following protein in E coli. Please select at least three different type of purification media and chromatographic conditions (buffer composition, pH, salt concentration etc) for rapid protein purification.

MADGDELLRV GTESDLNRIY NMVINVNTWG GAQMIEPISV GARAMDRAAG VRASHGSWAL YRQGSDDKAG YILRHTAEFI GELARTSLQS TASPAAVGNK RVRCFLYNNP DDAVGSVSIW REVLKTVGLS LAKEVGATSV LTKTRGIHIL MRGGDTRSID KVRIRAIEAI RLLTNYAIGL FALNRIDQCR DSNEMLSRVQ DFDTREFALA QKAREGADVN EKRIRRMVLS VANSDSPRPL GSRRHLMNLQ GLHQQITKVA LGRAQLYLLY DRVDEPEAPF WKRNNLKCWA AWSRCVSLLM KWTHESFAQR DRFMDMLNKR VEDSTADVPL APLNQHVTLL NSVKRKKKLS VSTGAPPRRR QRRKKRGAAG GHHHHHH-

* 3. We received the following plasmid sequence information from a customer. Please select a protein expression system and describe protocol for rapid and efficient protein purification.

(CMV Promoter sequence omitted)

GGATCC
GCC ACC
ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAAT TCC
gaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag gtcacatgcg
tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag tacaagtgca
aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa gccaaagggc
agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg accaagaacc
aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag caggggaacg
tcttctcatg ctccgtgatg cacgaggctc tgcacaacca ctacacgcag aagagcctct
ccctgtctcc gggtaaa

3C
ctg gag gtg ctg ttc cag ggc ccc

Target protein sequence (DNA sequence not shown)
EPPTACREKQYLINSQCCSLCQPGQKLVS DCTEFTETECLPCGESEFLDTWN RETHCHQHKYCDPNL GLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHR SC SPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWT SCETKDLVVQ QAGTNKTDVVCGPQDRLRRSSNTKVDKK VEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K


TAA
TCTAGA
CTCGAG
<BGH-pA>


* 4. An enzyme has the following properties

1. pI=10.1
2. N-6XHis Tag
3. Rapidly form aggregate at salt concentration less than 100 mM.
4. Lose activity at pH 5.0
5. Lose activity in the presence of detergents (even mild detergents)
6. Aggregate when trace amount of Ni or other divalent or trivalent metal ions is included in the eluent.

Please design a purification protocol to purify this protein to 95% purity with an extremely low endotoxin level and no significant loss of enzymatic activity

* 5. You are assigned to purify these proteins with only one set of columns:

1. Taq DNA polymerases
2. Thermolabile UDG
3. T4 DNA Ligase
4. T7 DNA Ligase
5. Klenow (3′→ 5′ exo-)
6. M-MuLV Reverse Transcriptase
7. T4 DNA Polymerase
8. T7 RNA Polymerase
9. Terminal deoxynucleotidyl Transferase (TdT)
10. Endonuclease VIII
11. Lambda Exonuclease
12. Thermolabile UDG
13. RNase Inhibitor
14. RNAse H
15. Benzonase
Please write a protein purification guideline.

* 6. Design and describe a novel E. Coli expression system for efficient and simplified purification of recombinant proteins without the need of column chromatography.

* 7. You are trying to purify a His tagged enzyme from 30 L E. coli culture. The protein is basic (pI9.5) and hydrophilic. What columns will you use to purify the protein after a Ni column? Design a scalable protein purification protocol to purify the protein to 99% purity.

* 8. Check the compatible expression plasmids and cell lines

  pGEX pET28b pTriEx pQE-TriSystem-6 pCDNA3.1 pFastBac Dual pQE-82L pBAD24 pFUSE-hIgG1-Fc
Top10
BL21
BL21(DE3)
BL21(DE3)pLysS
Origami 2(DE3)pLacI
Rosetta 2(DE3)pLysS
Tuner
Tuner(DE3)
Tuner(DE3)pLysS
Tuner(DE3)pLacI
Origami
Origami(DE3)
Origami(DE3)pLysS
Origami(DE3)pLacI
Origami B
Origami B(DE3)
Origami B(DE3)pLysS
Origami B(DE3)pLacI
Rosetta
Rosetta(DE3)
Rosetta(DE3)pLysS
Rosetta(DE3)pLacI
Rosetta 2
Rosetta 2(DE3)
Rosetta 2(DE3)pLysS
Rosetta 2(DE3)pLacI
RosettaBlue
RosettaBlue(DE3)
RosettaBlue(DE3)pLysS
RosettaBlue(DE3)pLacI
Rosetta-gami B
Rosetta-gami B(DE3)
Rosetta-gami B(DE3)pLysS
Rosetta-gami B(DE3)pLacI
Rosetta-gami 2
Rosetta-gami 2(DE3)
Rosetta-gami 2(DE3)pLysS
Rosetta-gami 2(DE3)pLacI
C41(DE3)
C43(DE3)
NiCo21(DE3)
Lemo21(DE3)
T7 Express lysY/Iq
BL21-AI
BL21(DE3)pLysE
BL21 Star™(DE3)
BL21 Star™ (DE3)pLysS
293F
CHO
Sf9/Sf21
Hela
M15
SG13009

* 9. Design and describe two expression systems, one for E coli, another for mammalian cells, from which recombinant proteins can be purified to 99% purity using only a single column, if a sufficient amount of proteins are expressed.

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