Protein Overexpression in E. coli and Purification by Affinity Chromatography 담당교수 : 이승택 교수님 담당조교 : 심현재

Introduction The feature of E. coli expression system Cultures of E. coli are easy and inexpensive. Many foreign proteins are expressed at high level. However, there is no post-translational modification (e.g., glycosylation or cleavage at specific site).

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

Introduction Two-step expression vector system ▪ E. coli BL21(DE3) target ▪ E. coli BL21(DE3) - Lac promoter (IPTG inducible) fused to T7 polymerase gene ▪ pET-32a(+) vector - T7 promoter fused to target cDNA - Target protein: His-tagged TEV protease (30 kDa) Target cDNA

Introduction Vector system pET-32a (+) vector map

Introduction Vector system pET-32a (+) vector map

Introduction Vector system pET-32a (+) vector map

Introduction Physical properties of proteins that can be applied for purification size hydrophobicity charge (isoelectric point) specific sequence feature (proline-rich sequence, affinity to metal ions, etc.) heat stability solubility

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.

Affinity chromatography Introduction Affinity chromatography 1. Separates according to their ability to ability to bind to a specific ligand that is connected to the beads of the resins. 2. Ligands are covalently attached to solid support. 3. The proteins that do not bind the ligand are washed through the column, the bound protein of interest is eluted by a solution containing free ligand. 4. One of the common forms of affinity chromatography presently used is the 6x-Histidine tag. Affinity chromatography handbook, Amersham biosciences

Introduction Hexa-Histidine (6x-his) tagged protein 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.

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

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.

Introduction Purification of 6x-his tagged protein using the Ni2+-NTA agarose

Introduction Preparation of cell extracts from bacteria bacterial cells removal of cell wall with lysozyme (optional) add protease inhibitors (optional) homogenization (sonication, french press, bead-beating) centrifugation Soluble protein Insoluble protein (inclusion bodies) - To prevent proteolytic cleavage and denaturation, it is important to carry out all these steps at low temperature.

Procedure < Day 1 > 1. Flask에 LB media 100 ㎖을 넣고 autoclave하여 보관한다. 2. 50 ㎖ tube에 LB media 5 ㎖, ampicillin(100 ㎎/㎖) 5 ㎕를 넣고 여기에 E. coli colony를 seeding한다. 3. E. coli를 37℃, 200 rpm으로 맞추어진 shaking incubator에서 overnight으로 키운다.

< Day 2 > E. coli 가 자란 것을 확인한다. 2. 전날 autoclave해둔 100 ㎖ LB media에 ampicillin(100 ㎎/㎖) 100 ㎕와 50 ㎖ tube에 서 자란 E. coli 1 ㎖ (1%) 을 inoculation 한다. 3. spectrophotometer로 600 ㎚에서 흡광도를 측정하여 O.D.값이 0.5가 될 때까지 키운다. (induction 전 시료 (생략): cell 1 ㎖ 을 harvest 한 후 DW 80 ㎕ 와 sample buffer 20 ㎕를 넣고 보관한다.) 4. O.D.가 0.5가 되면 1M IPTG 10 ㎕를 각 flask에 넣어주어 25℃, 100 rpm으로 induction한다. 5. 8시간 동안 더 키운다. (induction 후 시료 (생략): cell 1 ㎖ 을 harvest 한 후 DW 80 ㎕ 와 sample buffer 20 ㎕ 를 넣고 보관한다.) 6. 50 ㎖ tube 에 옮겨서 4,000 rpm, 10 min centrifuge하여 E. coli를 모으고 -20 ℃에서 보관한다.

< Day 3 > 1. Resuspending of cells 얼어있는 cell pellet에 lysis buffer를 cell culture 부피의 1 / 10 을 가해 resuspend 한다. 100 ㎖ culture한 cell pellet이므로 10 ㎖ lysis buffer로 resuspend함. (lysis buffer 조성: 20 mM Sodium Phosphate (pH 7.6), 300 mM NaCl , 2 mM β-mercaptanol, 0.1% TritonX-100, 10 mM imidazole) 2. Lysis of cells  resuspending 한 sample중 10 ㎖ 을 50 ㎖ tube에서 sonication으로 cell을 파쇄한다. (30 sec burst, 90 sec cooling, 5 times) # sonication 시, 거품이 나지 않도록 rod가 sample의 중간에 오도록 한다. - cell lysate : cell lysis 한 sample 10 ㎖ 중에서 10 ㎕ 을 취하여 sample buffer 2.5 ㎕ 가 들어있는 tube 에 넣는다. 3. Separation of soluble proteins high speed centrifuge로 7,000 rpm , 4℃, 10 min centrifuge 하여 supernatant를 얻는다. Soluble 시료: Supernatant중 sample 10 ㎕ 를 취하여 sample buffer 2.5 ㎕ 가 들어있는 튜브에 넣는다. Insoluble 시료(생략): precipitate를 urea buffer 10 ㎖ 로 분산하여, sample 10 ㎕ 를 취하여 sample buffer 2.5㎕ 가 들어있는 튜브에 넣는다.

4. Ni2+- NTA agarose affinity column chromatography Ni2+-NTA resin binding buffer  : 10 mM imidazole, lysis buffer (pH 7.6)  washing buffer : 40 mM imidazole, lysis buffer (pH 7.6)   elution buffer : 100 mM or 200 mM imidazole, lysis buffer (pH 7.6)  1) 20 % ethanol 에 있는 resin을 resuspention하여 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 protein을 loading한다. - FT 시료: flow-through 중 10 ㎕ 를 취하여 sample buffer 2.5 ㎕ 가 들어있는 튜브에 넣는다. 5) bed volume(200 ㎕) 10배의 washing buffer를 3번으로 나누어 흘려준다. - W1,2,3 시료: washing 시료 중 10 ㎕를 취하여 sample buffer 2.5 ㎕가 들어있는 튜브에 넣는다.(W1, 2 생략) 6) bed volume(200 ㎕) 2배의 elution buffer를 fraction별로 흘려준다. - E1, E2 , E3, E4, E5 시료: elution의 각 fraction 별 sample 10 ㎕ 를 취하여 sample buffer 2.5 ㎕ 가 들어있는 튜브에 넣는다. 남은 sample은 얼음에서 보관한다.

5. SDS-PAGE 확인 1) 12.5% SDS-PAG을 만든다.    2) sample 10 ㎕에 sample buffer 2.5 ㎕를 넣은 tube를 3 min동안 끓인다.    3) sample을 loading한다. cell lysate/soluble 시료/FT 시료  6.25 ㎕ (시료 기준 5 ㎕) 씩 W3 시료/E1, E2, E3, E4, E5 시료    4) 80 V에서 30 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 뺀 후 말린다. # 조교가 destaining된 gel을 scan하여 생화학과 홈페이지에 게시 예정.

Procedure # Ni2+- NTA agarose affinity column regeneration     다음 solution들을 차례대로 흘려준다.     1) 6 M guanidine-HCl, 0.2 M acetic acid 2 volume     2) water 5 volume     3) 2% SDS 1 volume     4) 25%, 50%, 75% ethanol 1 volume     5) 100% ethanol 5 volume     6) 75%, 50%, 25% ethanol 1 volume     7) water 1 volume     8) 100 mM EDTA, pH 8.0 5 volume     9) water 5 volume 10) recharge with 100 mM NiSO4 2 volume 11) water 5 volume   12) 20% ethanol 1 volume으로 suspension하여 column에서 빼내고 4℃에서 보관

보고서 작성시 필수적으로 포함되어야 할 사항 (선행 실험의 결과 참고) Purification yield 계산하시오 . Purity 및 Purification fold 계산하시오. 대상 단백질 10 mg을 얻어야 한다면 대상 단백질을 발현하는 E. coli를 LB media에서 얼마만큼 배양해야 할 것인지 추정하시오.

Results from the preliminary experiment Fraction number of Elution Washing (1/140) 40 mM imidazole Cell lysate (1/3000) Soluble 시료 (1/3000) 100 mM Imidazole (1/80) 200 mM Imidazole (1/80) FT 시료 (1/3000) W1 W2 W3 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12

Results from the preliminary result Starting material (1번 cell lysate)을 기준으로 하여 정제 step별 purification fold 및 yield의 계산 Lane Sample Total volume of step (ml) Gel loading volume (ul) rate Target protein in gel (ug) Target protein in step (ug) Total protein in gel (ug) Total protein in step (ug) Purity of 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 Cell lysate 15 5 3,000 0.60 1,800 13.50 40,500 0.044 　1.000 　1 2 Soluble 시료 12.78 38,340 0.047 1.068 3 FT 시료 0.16 482 11.57 34,698 4 E1 0.4 80 0.58 47 2.17 174 E2 1.63 131 3.93 314 6 E3 1.28 102 2.36 189 7 E4 1.32 106 1.69 135 8 E5 1.31 105 1.68 134 9 E6 2.21 177 2.29 183 10 E7 2.10 168 2.20 176 11 E8 0.90 72 1.00 12 E9 0.26 21 0.33 26 13 E10 0.12 0.55 44 14 E11 0.06 0.13 E12 0.04 *** Elution (pool) 946 1,475 0.641 14.568 0.526