Protein Expression & Purification 3

* 1. Write a simplified protocol for the purification and on-column refolding of an insoluble histidine-tagged protein from an 8 L E. coli culture using HisTrap FF 20 ml with ÄKTAprime plus

* 2. Describe in your words: strategy for successful purification of a target protein by gel filtration

* 3. Describe in your words: strategy for successful purification of a target protein by anion exchange chromatography

* 4. Design a purification protocol for 6X His tagged restriction enzymes based on the following tables.
Columns:
1. 10 ml Ni resin
2. 15 ml phosphocellulose
3. 15 ml heparin agarose
4. 30 ml Sephadex G25

Salt concentration for elution from absorbent

Salt concentration for elution from absorbent

* 5. Design an efficient purification protocol for this protein using 100 ml Q FF column and 25 ml SP HP column. Ni resin can not be used because trace amount of Ni ion leaked from the resin will promote protein aggregation. Protein solution should not be diluted as protein will lose activity when it is in diluted solution. Protein will form aggregate when concentration is > 15 mg/ml. Desalting column is also not available.

Hint: Use this website to calculate binding strength to ion exchange resin:
http://www.innovagen.se/custom-peptide-synthesis/peptide-property-calculator/peptide-property-calculator.asp

DNA sequence:

CCATGG GCCATCACCATCACCATCACGGCATGGGCGCTGCAGGTCGCAAGAAACGTCGCCAACGTCGCCGTCCGCCTGCAGGCACTAGTGTAAGCTTGAAGAAGAAGAGGAAGGTGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATTAG
CTCGAG CAC CAC CAC CAC CAC CAC TGA

* 6. You have finally purified a protein. When you run this protein on Superose 12 10/300 GL you got a peak of a molecular weight of 240 kDa. However when you added 6M guanidine hydrochloride to the mobile phase (buffer) you saw only one peak of 60 kDa. When you run a SDS-PAGE gel you saw two bands (34 kDa and 26 kDa). Explain why.

* 7. For gel filtration chromatography
1. Optimal salt condition for gel filtration on Superdex 200 prep grade XK 16/60 is __
2. Protein concentration should be _ mg/ml, flow rate should be _
3. When protein is in a buffer containing 50% glycerol or 8M urea, you can inject 5 ml. True or False
4. When working with a new sample, the buffer composition should be _
5. When sample in 8M urea or guanidine hydrochloride, you should use
5.1. Superdex
5.2. Sephacryl
5.3. Sepharose
5.4. Sephadex
6. Higher flow rate is better for separation (resolution) because it reduces sample diffusion, True or False
7. Inject 2.5 ml of 5 mg/ml sample to Superdex 200 prep grade XK 16/60 is better than injecting 5 ml of 2.5 mg/ml sample, True or False
8. You have optimize gel filtration condition using HiLoad 16/60 Superdex 200 pg. To scale up production you can
8.1 Use HiLoad 26/60 Superdex 200 pg, True or False
8.2 Connecting two HiLoad 16/60 Superdex 200 pg in series. True or False

* 8. You have partially purified the following two proteins using Ni resin. You tried to concentrate these two proteins using spin concentrators before loading to next column. You accidentally mis-labeled the concentrator tubes and two proteins have been mixed. Please design a purification protocol to purify these two proteins from the mixture.

1.
MGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPD
IIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYN
KDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDI
KDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTS
KVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKP
LGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD
EALKDAQTNSSSNNNNNNNNNNLGDDDDKLEVLFQGPHMEGMLKGEGPGPLPPLLQQYVE
LRDQYPDYLLLFQVGDFYECFGEDAERLARALGLVLTHKTSKDFTTPMAGIPLRAFEAYA
ERLLKMGFRLAVADQVEPAEEAEGLVRREVTQLLTPGTLLQESLLPREANYLAAIATGDG
WGLAFLDVSTGEFKGTVLKSKSALYDELFRHRPAEVLLAPELLENGAFLDEFRKRFPVML
SEAPFEPEGEGPLALRRARGALLAYAQRTQGGALSLQPFRFYDPGAFMRLPEATLRALEV
FEPLRGQDTLFSVLDETRTAPGRRLLQSWLRHPLLDRGPLEARLDRVEGFVREGALREGV
RRLLYRLADLERLATRLELGRASPKDLGALRRSLQILPELRALLGEEVGLPDLSPLKEEL
EAALVEDPPLKVSEGGLIREGYDPDLDALRAAHREGVAYFLELEERERERTGIPTLKVGY
NAVFGYYLEVTRPYYERVPKEYRPVQTLKDRQRYTLPEMKEKEREVYRLEALIRRREEEV
FLEVRERAKRQAEALREAARILAELDVYAALAEVAVRYGYVRPRFGDRLQIRAGRHPVVE
RRTEFVPNDLEMAHELVLITGPNMAGKSTFLRQTALIALLAQVGSFVPAEEAHLPLFDGI
YTRIGASDDLAGGKSTFMVEMEEVALILKEATENSLVLLDEVGRGTSSLDGVAIATAVAE
ALHERRAYTLFATHYFELTALGLPRLKNLHVAAREEAGGLVFYHQVLPGPASKSYGVEVA
AMAGLPKEVVARARALLQAMAARREGALDAVLERLLALDPDRLTPLEALRLLQELKALAL
GAPLDTMKGKLAAALEHHHHHH-

2.
MGHHHHHHMAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAAELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI

* 9. You are loading 200 ml of cleared lysate (supernatant from ultracentrifuge) to Ni column, you notice that the flow become slower and slower after loading ~50 ml of lysate. You see white aggregates are stacking on top of the resin. What will you do?
Buffer for lysing E coli: 50 mM Tris-HCl, pH 8.0 at 25 oC, 500 mM NaCl, 1 mM beta-ME, 25 mM imidazole.
Append your name after this question.