Downstream Processing Lecture 7 ISAT 451, Fall 2003 College of Integrated Science and Technology James Madison University

Gene Discovery Cloning & Transformation Transformation(유용 유전자) Research & Development Upstream Processes Cell Line Development Media Preparation(세포주개발 , 배지확립) Microbial Fermentation Mammalian Cell Culture(배양) Production Harvest Cells(세포수확) Cell Disruption(분쇄) Protein Purification(단백질 분리) Downstream Processes Product Analytical Tests(분석)

Harvest Cells Centrifugation Filtration(원심분리, 여과) Growth Medium Cell Disruption Enzymatic Chemical Physical Cell Debris Total Proteins Unwanted Proteins Purification Steps Pure Protein Analytical Tests

Centrifugation(원심분리) When a centrifugal force is applied to an aqueous mixture, components of larger size and density will sediment faster(크기, 밀도로 분리) Low speed centrifugation is used to separate intact cells from spent medium(세포분리) High speed centrifugation can be used to separate subcellular components(소기관분리)

Fixed-Angle Centrifugation *축이 고정된 원심분리기 이용

Swinging-Arm Centrifugation

Differential Centrifugation (밀도차이 원심분리)

Filtration(여과) Filtration can be used to separate cells from growth medium or subcellular components(세포와 배지분리) To harvest cells, a pore size from 0.2 to 0.45 micrometers is used A dead-end or cross-flow filtration system can be used to harvest cells

Dead-End Filtration A membrane with pores smaller than cells is used to trap cells. As cells are trapped on the membrane, the flow rate decreases.

Cross-Flow Filtration

Cell Disruption(분쇄) Chemical: alkali, organic solvents, detergents Enzymatic: lysozyme, glucanases, chitinase Physical: osmotic shock, freeze/thaw Mechanical: sonication, homogenization, wet milling, French press

Chemical Disruption Detergents such as Trition X-100 or NP40 can permeabilize cells by solubilizing membranes.(계면활성제이용-단백질변성의 위험) Detergents can be expensive, denature proteins, and must be removed after disruption

French Press Cells are placed in a stainless steel container. A tight fitting piston is inserted and high pressures are applied to force cells through a small hole. (압력차에 의한 분쇄)

Homogenization Cells are placed in a closed vessel (usually glass). A tight fitting plunger is inserted and rotated with a downward force. Cells are disrupted as they pass between the plunger and vessel wall.

Sonication(초음파분쇄) A sonicator can be immersed directly into a cell suspension. The sonicator is vibrated and high frequency sound waves disrupt cells.

Disrupted Cells Cell Lysate Pellet (discard) Centrifuge Supernatant Cell-Free Lysate Proteins, Nucleic Acids, Small Molecules Unwanted Molecules Multiple Purification Steps Pure Protein

Protein Purification Techniques Centrifugation Filtration Precipitation Chromatography Electrophoresis Note: some techniques are applicable for small-scale (analytical purposes) while some can be scaled-up for preparative use.

Principles of Protein Purification Separation of proteins is a function of the physical properties that make proteins different, in particular:(단백질분리는 다음의 특성이 다른 단백질의 물리적인 특성에 따라서 실행한다) Size Shape Charge (local and net) Hydrophobicity Solubility Affinity

Precipitation(침전) Precipitation is caused by changes that disrupt the solvating properties of water(침전은 물의 용매로서의 성질을 파괴하는 변화에 의해 일어난다) Changes in pH, ionic strength, temperature, and the addition of solvents can cause precipitation (loss of solubility)(pH, ionic strength, 온도 및 용매의 첨가에 의한 변화가 침전을 유도한다, 즉 용해도의 상실로) Most proteins have a unique set of conditions that result in precipitation 대부분의 단백질은 침전되는 특별한 조건을 갖는다)

Precipitation with Salt In practice, most procedures use the salt ammonium sulfate (NH4)2SO4 to precipitate proteins(실험적으로 단백질 침전 시 (NH4)2SO4 를 사용한다) The amount of salt required is directly related to the number and distribution of charged and nonionic polar amino acids exposed on the surface of the protein(필요한 염의 양은 단백질표면의 전하를 띠거나 극성아미노산의 수와 분포에 직접 관련된다)

Chromatography Ion Exchange Gel Filtration (size-exclusion) Hydrophobic (reverse phase) HPLC (High Performance Liquid Chromatography) Affinity Chromatofocusing (isoelectric focusing)

Column Chromatography

Chromatography Resins Exchange Group Counterion (salts) Solid Support + or - Cellulose Dextrans Agarose DEAE (+) CM (-) NaCl

Ion Exchange Chromatography(이온교환수지 크로마토그래피) A charged portion of a protein will bind to exchange groups of opposite charge.(단백질의 전하 띤 부위가 반대전하를 갖는 교환수지에 결합한다) Increasing concentrations of salt will elute bound proteins(염농도를 증가시키면 결합된 단백질이 떨어져 나온다).

Gel Filtration Chromatography Proteins that enter porous beads will migrate slower than proteins that are excluded from the pores.(다공성 구술에 들어간 단백질은 그렇지 않은 것보다 천천히 이동) Separation is a function of relative size and shape(분리는 상대적인 크기와 모양에 함수)

Gel Filtration Chromatography Vo = Void volume (the excluded volume surrounding the beads) Vt = Total volume (the excluded volume plus the internal volume of the beads) Ve = Intermediate volume (partially excluded) Kav = Partition Coefficient

Gel Filtration Chromatography Kav = Ve - Vo / Vt – Vo 1.0 Kav or Mol Wt Log Mol Wt or Volume

Affinity Chromatography(선택적결합 크로마토그래피) A ligand that specifically binds to a unique protein is linked to a solidsupport.(특정단백질에 결합하는 리갠드를 고체형에 결합시킴) Specific proteins bind to the ligand while other proteins pass through. The protein can be eluted by salts or changes in pH.(특정단백질을 그 리갠드에 결합시키고, 염이나 pH를 변화시켜 떨어트린다)

Affinity Chromatography Solid Support Ligand Specificity Cellulose Sepharose DNA IgG Histones Protein A

Hydrophobic Chromatography Polar Group Solid Support Elution Sepharose Phenyl Octyl Low salt Proteins are bound in high salt. The salt concentration is lowered to elute. Binding is a function of the hydrophobic amino acids exposed on the surface.(단백질은 높은 염존재 하에서 결합하고, 염농도가 낮아지면 떨어진다. 결합력은 그 표면의 소수성 아미노산의 함수이다)

Isoelectric Focusing For any protein, there is a characteristic pH at which the protein has no net charge (isoelectric point). (단백질이 전하값이 제로가 되는 특정 pH를 Isoelectric point, 즉 pI 라함) At the isoelectric pH, the protein will not migrate in an electric field.(그 pH에서, 단백질은 전기장에서 이동하지 않는다)