Transcript Centrifugation(원심분리)

Downstream Processing
Lecture 7
ISAT 451, Fall 2003
College of Integrated Science and
Technology
James Madison University
Research & Development
Upstream Processes
Production
Downstream Processes
Product
Gene Discovery
Cloning & Transformation
Transformation(유용 유전자)
Cell Line Development
Media Preparation(세포주개발
, 배지확립)
Microbial Fermentation
Mammalian Cell Culture(배양)
Harvest Cells(세포수확)
Cell Disruption(분쇄)
Protein Purification(단백질
분리)
Analytical Tests(분석)
Harvest Cells
Growth Medium
Cell Disruption
Cell Debris
Total Proteins
Unwanted Proteins
Centrifugation
Filtration(원심분리,
여과)
Enzymatic
Chemical
Physical
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.(계면활성제이
용-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
Pellet
(discard)
Cell Lysate
Centrifuge
Supernatant
Unwanted
Molecules
Cell-Free Lysate
Proteins, Nucleic Acids,
Small 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
Solid Support
Exchange
Group
Counterion
(salts)
+ 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
Solid Support
Sepharose
Sepharose
Polar
Group
Elution
Phenyl
Octyl
Low salt
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)