Protein Overexpression in E Protein Overexpression in E. coli and Purification by Affinity Chromatography Prof. Seung-taek Lee Teaching Assistant Yang-chae Kim In-jung Hwang

1. Introduction – The feature of E. coli expression system The expression of proteins in Escherichia coli(E. coli) is the easiest, quickest and cheapest method. There are many commercial and non-commercial expression vectors available with different N- and C-terminal tags and many different strains which are optimized for special applications. Many foreign proteins are expressed at high level. However, there is no post-translational modification (e.g., glycosylation or cleavage at specific site). Protein overexpression system E.coli Prokaryotic cell Yeast Insect cell Eukaryotic cell mammalian

1. Introduction - lac operon Regulation - Lactose binding of lac repressor to operator lac operon turn off - Lactose No binding lac operon turn on IPTG; a lactose analog that blocks lac repressor

1. Introduction – Two-step expression vector system ▪ E. coli BL21(DE3) - Lac promoter (IPTG inducible) fused to T7 polymerase gene ▪ pET vector - T7 promoter fused to target cDNA - Target protein: His-tagged Tev protease (27 kDa) The pET expression system LacI represses the lac promoter. T7 RNA polymerase is transcribed when IPTG binds and triggers the release of tetrameric LacI from the lac operator. Transcription of the target gene from the T7 promoter is subsequently initiated by T7 RNA polymerase. T7 promoter is a 20-nucleotide sequence not recognized by the E.coli RNA polymerase.

1. Introduction – Vector system 1. Moffatt, B.A. and Studier, F.W. (1986) J. Mol. Biol. 189, 113ó130. 2. Rosenberg, A.H., Lade, B.N., Chui, D., Lin, S., Dunn, J.J., and Studier, F.W. (1987) Gene 56, 125-135.

1. Introduction – pET-15b cloning / expression region

1. Introduction – Physical properties of proteins that 1. Introduction – Physical properties of proteins that can be applied for purification specific sequence feature (proline-rich sequence, affinity to metal ions, etc.) Size hydrophobicity charge

1. Introduction – Important matters to consider before 1. Introduction – Important matters to consider before starting the purification Sample and target protein properties Strategy Temperature stability Use low temp. pH stability Selection of buffers Detergent requirement Consider choice of detergents Co-factor for stability or activity Select additives, salt, etc.

1. Introduction – Affinity chromatography Affinity chromatography separates according to their ability to bind to a specific ligand that is connected to the beads. 2. The proteins that do not bind the ligand are washed through the column. 3. The bound protein of interest is eluted by a solution containing free ligand. * One of the common forms of affinity chromatography presently used is the 6x Histidine tag. Affinity chromatography handbook, Amersham biosciences

1. Introduction – Hexa-Histidine (6x-his) tagged protein - To easily purify a protein by affinity column chromatography, often add Hexa-histidine tag to the protein by recombinant DNA technology. - 6x-his tagged protein is the Hexa-Histidine peptide tagged in N- or C-terminal end of target protein. - 6x-his tag is small, uncharged, and does not generally affect folding of the fusion protein within the cell. - NTA (nitrilotriacetic acid) agarose occupies four of the six ligand binding site in the coordination sphere of the nickel ion, leaving two sites free to interact with the 6x-his tag.

1. Introduction – Interaction between neighboring residues in the 1. Introduction – Interaction between neighboring residues in the 6x-his tag and Ni2+-NTA matrix

1. Introduction – 6x-his tagged protein - Imidazole ring is part of the structure of histidine and bind to nickel ions. - At low imidazole concentration, nonspecific, low affinity binding of background protein is prevented. - At high imidazole concentration, 6x-his tagged proteins are eluted. - Ni2+-NTA agarose is sufficient for the binding of approximately 5-10 mg of 6x-his tagged protein per milliliter of resin.

1. Introduction – Preparation of cell extracts from bacteria bacterial cells removal of cell wall with lysozyme (optional) Cell disruption (Sonication, homogenization, french press or bead-beating) centrifugation Soluble protein Insoluble protein (inclusion bodies) add protease inhibitors (optional) - To prevent proteolytic cleavage and denaturation, it is important to carry out all these steps at low temperature. - The target protein can be purified from either soluble fraction or insoluble inclusion bodies

1. Introduction – Purification of 6x-his tagged soluble protein using 1. Introduction – Purification of 6x-his tagged soluble protein using the Ni2+-NTA agarose Qiagen Ni-NTA Agrose product detail http://www.qiagen.com/us/products/catalog/sample-technologies/protein-sample-technologies/purification-kits-and-resins/ni-nta-agarose/#productdetails

2. Procedure < Day 1 > 15 ㎖ tube에 LB media 3 ㎖을 넣고 autoclave하여 보관한다. 2. 15 ㎖ tube에 LB media 3 ㎖, ampicillin(100 ㎎/㎖) 3 ㎕를 넣고 여기에 E. coli colony를 seeding한다. 3. E. coli를 37 ℃, 200 rpm으로 맞추어진 shaking incubator에서 overnight으로 키운다.

2. Procedure < Day 2 > 1. E. coli 가 자란 것을 확인한다. 2. autoclave해둔 10 ㎖ LB media에 ampicillin (100 ㎎/㎖) 10 ㎕를 가하고, overnight으로 배양한 E. coli 100 ㎕ (1%)을 inoculation 한다. 3. spectrophotometer로 600 ㎚에서 흡광도를 측정하여 O.D.값이 0.5가 될 때까지 키운다. 4. O.D.가 0.5가 되면 1 M IPTG 10 ㎕를 tube에 넣어주어 25℃, 100 rpm으로 8시간 동안 더 키워서 target 단백질을 induction한다. 5. 50 ㎖ tube를 4,000 rpm, 10 min centrifuge하여 E. coli를 모은다.

2. Procedure 1. Resuspending of cells cell pellet에 10 mM imidazole을 포함하는 lysis buffer를 cell culture 부피의 1 / 10 을 가해 resuspend 한다. 10 ㎖ culture한 cell pellet이므로 1 ㎖로 resuspend함. (lysis buffer 조성: 25 mM Sodium Phosphate (pH 7.5), 100 mM NaCl , 2 mM β-mercaptoethanol) 2. Lysis of cells  resuspending 한 sample을 microtube에 옮긴다. 이 시료를 sonication으로 cell을 파쇄한다. (30 sec burst, 90 sec cooling, 5-8 times) - 주의! sonication 시, 거품이 나지 않도록 rod가 sample의 중간에 오도록 한다. 3. Separation of soluble proteins microtube centrifuge로 14,000 rpm, 4℃, 3 min centrifuge 하여 supernatant를 얻는다. (Input 시료 : Supernatant 중 20 ㎕를 취하여 5X sample buffer 5 ㎕ 가 들어있는 tube 에 넣는다. )

2. Procedure 4. Ni2+- NTA agarose affinity column chromatography <각 단계 buffer 조성> binding buffer  : 10 mM imidazole이 포함된 lysis buffer (pH 7.5)  washing buffer : 30 mM imidazole이 포함된 lysis buffer (pH 7.5)   elution buffer : 500mM imidazole이 포함된 lysis buffer (pH 7.5)  1) Binding buffer 에 equilibrium 되어있는 resin (200 ㎕)을 흔들어준 후 polypropylene column에 붓는다. 2) resin이 가라앉으면 binding buffer 를 흘려주어 column bed 바로 위까지 비운다. 이때 resin이 마르지 않도록 주의한다. 3) bed volume(200 ㎕)의 3배의 binding buffer를 흘려준다. 4) Soluble protein이 들어있는 cell lysate를 loading하고, flow through를 받는다. F.T.를 다시 loading하고 F.T.를 받는 과정을 4회 반복한다. (최종적으로 받은 F.T.는 총 5회의 F.T.를 진행한 것이어야 함. ) ( FT 시료 : 최종 flow-through 중 20 ㎕을 취하여 5X sample buffer 5 ㎕가 들어있는 tube에 넣는다.) 5) bed volume(200 ㎕) 9배의 washing buffer를 흘려준다. (3배의 Bed volume X 3회 : W1, W2, W3) ( W3 시료 : W3 시료 중 24 ㎕를 취하여 5X sample buffer 6 ㎕ 들어있는 tube 에 넣는다.) 6) bed volume(200 ㎕) 10배의 elution buffer를 400 ㎕씩 5번에 나누어 흘려주어 column을 지난 elution fraction을 받는다. (E1, E2 , E3, E4, E5 시료 : elution의 각 fraction 별 sample 16 ㎕ 를 취하여 5X sample buffer 4 ㎕가 들어있는 tube 에 넣는다.)

2. Procedure 5. SDS-PAGE 확인 1) 12.5% SDS-PAGE 을 만든다.    2) sample에 sample buffer를 가하여 준비한 microtube를 4 min동안 끓인다.    3) sample을 loading한다.    4) 80 V에서 10 min동안 초기 running 한다. 5) 130 V 에서 dye가 끝에 올 때까지 running한다.    5) Coomassie brilliant blue (staining solution)로 30 min동안 staining한다.    6) destaining solution에 넣고 dye를 제거한다. - destaining된 gel을 scan하여 생화학과 홈페이지에 게시 예정.

3. Results 25kDa 35kDa input 1/50 FT 1/50 Wash2 1/25 Elution1 1/25

Table: Analysis of the result from the purification process Lane Sample Total volume of the step (ul) Gel loading volume (ul) Ratio of loading volume out of total volume Target protein in gel (ug) Target Protein in step Total Protein Protein in Step Purity each step (target protein/ total protein) Purification fold (purity of each step /purity of the starting material) Yield (target protein in each step/target protein in the starting material) 1 Input 1000 20 50 2.1 105 14.21 710.5 0.15 1.00 2 F.T 0.1 5 11.41 570.5 3 Wash 3 600 24 25 0.002 0.05 4 E1 400 16 1.9 47.5 52.5 0.89 5.98 0.55 E2 0.4 10 0.5 12.5 6 E3 0.001 0.025 7 E4 8 E5

3. Homework 보고서 작성시 필수적으로 포함되어야 할 사항 (선행 실험의 결과 참고) 예시된 표와 같이 purification yield, purity 및 purification fold를 계산하여 표로 작성하시오. 단백질 정제과정에서 purification yield와 purification fold에서 얻을 수 있는 정보를 설명하시오.