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

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; a lactose analog that 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 vector - T7 promoter fused to target cDNA - Target protein: His-tagged Tev protease (27kDa) Target cDNA

Introduction Vector system

pET-15b cloning/expression region

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.)

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. 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

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.

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.

(Sonication, homogenization, french press or bead-beating) 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

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

Procedure < Day 1 > 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.

< Day 2 > 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 대상 단백질을 induction한다. 5. 50 ㎖ tube를 4,000 rpm, 10 min centrifuge하여 E. coli를 모은다.

# sonication 시, 거품이 나지 않도록 rod가 sample의 중간에 오도록 한다. < Day 3 > 1. Resuspending of cells cell pellet에 lysis buffer를 cell culture 부피의 1 / 10 을 가해 resuspend 한다. 10 ㎖ culture한 cell pellet이므로 1 ㎖ lysis buffer로 resuspend함. (lysis buffer 조성: 25 mM Sodium Phosphate (pH 7.5), 100 mM NaCl , 2 mM β-mercaptanol, 10 mM imidazole) 2. Lysis of cells  resuspending 한 sample을 microtube에 옮긴다. 이 시료를 sonication으로 cell을 파쇄한다. (30 sec burst, 90 sec cooling, 5-8 times) # 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. Soluble 시료: Supernatant중 sample 20 ㎕ 를 취하여 4X sample buffer 6.7 ㎕ 가 들어있는 tube 에 넣는다. Insoluble 시료(생략): precipitate를 urea buffer 1 ㎖ 로 분산하여, sample 10 ㎕ 를 취하여 sample buffer 2.5㎕ 가 들어있는 튜브에 넣는다. (0) (X)

4. Ni2+- NTA agarose affinity column chromatography Ni2+-NTA resin binding buffer  : 10 mM imidazole, lysis buffer (pH 7.5)  washing buffer : 30 mM imidazole, lysis buffer (pH 7.5)   elution buffer : 500mM imidazole, 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 protein을 loading한다. - FT 시료: flow-through 중 20 ㎕ 을 취하여 4X sample buffer 6.7 ㎕들어있는 tube 에 넣는다. 5) bed volume(200 ㎕) 9배의 washing buffer를 흘려준다. (3배의 Bed volume X 3회 : W1, W2, W3) - W3 시료: W3 시료 중 10 ㎕를 취하여 4X sample buffer 3.3㎕ 들어있는 tube 에 넣는다. 6) bed volume(200 ㎕) 10배의 elution buffer를 fraction별로 흘려준다. 이때는 400 ㎕씩 5번에 나누어 받는다. - E1, E2 , E3, E4, E5 시료: elution의 각 fraction 별 sample 10 ㎕ 를 취하여 4X sample buffer 3.3 ㎕가 들어있는 tube 에 넣는다.

5. SDS-PAGE 확인 1) 12.5% SDS-PAGE 을 만든다.    2) sample에 sample buffer를 가하여 준비한 microtube를 5 min동안 끓인다.    3) sample을 loading한다. soluble 시료/FT 시료  20 ㎕ 씩 Wash3, E1, E2, E3, E4, E5 시료  10 ㎕ 씩    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. # 조교가 destaining된 gel을 scan하여 생화학과 홈페이지에 게시 예정.

A result from the experiment (per 10 ml culture) 170 130 100 70 55 40 35 25 15 Input 1/50 FT 1/50 W3 1/60 E1 1/40 E2 1/40 E3 1/40 E4 1/40 E5 1/40 TEV Protease 27kD

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 2.6 130 120.4 6020 0.02 2 F.T 0.1 5 116.8 5800 3 Wash 3 600 10 60 0.03 1.8 4 E1 400 40 2.0 80 2.1 84 116/122 =0.95 0.95/0.02 =47.5 116/130 =0.89 E2 0.8 32 0.85 34 6 E3 7 E4 8 E5 * Concentration of total protein in each sample was measured by Bradford assay.

보고서 작성시 필수적으로 포함되어야 할 사항 (선행 실험의 결과 참고) 대상 단백질 10 mg을 얻어야 한다면 대상 단백질을 발현하는 E. coli를 LB media에서 얼마만큼 배양해야 할 것인지 추정하시오. 예시된 표와 같이 purification yield, purity 및 purification fold를 계산하시오. 단백질 정제과정에서 purification yield와 purification fold에서 얻을수 있는 정보를 설명하시오.