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- 유전공학특론 - In Vitro Expression. Expressway TM In Vitro Protein Synthesis System. Lab. of Microbial Ecology Kwon Kyung-A. Introduction. Coupled Transcription-Translation in Prokaryotic Cell-Free System.

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-유전공학특론- In Vitro Expression

ExpresswayTM

In Vitro Protein Synthesis System

Lab. of Microbial Ecology

Kwon Kyung-A


Introduction

Coupled Transcription-Translation

in Prokaryotic Cell-Free System

-Cell-free protein synthesis in a coupled transcription-translation system은 endogenous nuclease에 의한 mRNA의 degradation 을 줄일 수 있고 initiation site 가까이에mRNA secondary structure가 형성되는 것을 막을 수 있다.

-Two such system have found the widest application

*E.coli S30 Extract system(devised by Zubay)

*The fractionated system(of Gold and Schweiger)


The cell-free System of Zubay

-Reagents(The water with diethylpyrocarbonate to inactivate RNase activity )

-Preparation of Cells (E.coli S30, MRE600..)

-Preparation of the S30 Extract

-Optimisation of the system

The cell-free System of Gold and Schweiger

-Reagents(The water with diethylpyrocarbonate to inactivate RNase activity )

-Preparation of Cells (E.coli..)

-Preparation of cell-free Extract(Elute the protein from the DEAE-cellulose column)

-Optimisation of the system


Advantages and disadvantages of the Zubay System

-The system is faily easy to prepare and extremely easy to use.

-The S30 is a crude extract and therefore most regulatory factors should be present.

-the S30 extract can be stored in liquid nitrogen for several years

-The extract may contain some residual DNA which can serve as template to give a wide range of background polypeptides

-The S30 extract contains membrane fragments,


Advantages and disadvantages of the Gold and Schweiger System

-The DEAE-cellulose fractionation makes the system very low residual DNA

-The Gold and Schweiger system probably contains lower concentrations of endogenous amino acid. (incorporaion of radioactive amino acids)

-The system is complicated to prepare and use

-The protein fraction and ribosomes are stable for only a few months

-Certain protein factors may be lost


Introduction System

The ExpresswayTM Mechanism

-E.coli S30 extract

(contianing the cellular machinery required to drive a coupled transcription and translation reaction)

-reaction buffer

-unique ATP regenerating system for energy

-required amino acid

-T7 RNA polymerase

-gene of interest incubation for 2h


Expressway System System의 장점

Time consuming transformation, cell maintenance, expression opimization step

Intact cell을 사용해서 protein 을 생산하는 전통적인 방법보다 편리하다

2시간 안에 Coomassie-stained gel에서 관찰되는 충분한 protein을 얻을 수 있다.

결과를 빨리 확인할 수 있으므로 응용범위가 넓다,

protein characterization, verification and functional analysis, production of toxic proteins, and high-throughput screening.


High protein yeilds with Expressway SystemTM System

PCR

  • 2.5 ul of each reaction

  • Total protein yeild ([35S]-methionine incorporation assay)-size of the protein, sequence of the gene of interest, quality of the DNA에 따라 protein yeild에 차이가 남.


Expressway SystemTM system offers your choice of an optimized two vector

  • Gateway Technology를 사용해서 in vitro expression에서 high level이고 gene을 쉽게 cloning 할 수 있는 vector를 찾아냄

  • pEXP1-DESTTM

  • pEXP2-DESTTM

Advantage of Gateway-compatible vector

  • Easy recombinational cloning

  • Nearly limitless expression options in other host system


pEXP1-DEST SystemTM

Gene of interest의 N-terminal 에

epitope와 purification seq. 를 붙임

size 4.6 kb

pUC origin(high-copy number)

Ampicillin resistance gene

T7 promoter, T7 terminator

RBS(ribosome binding site)

ATG(start codon)

6xHis (rapid purification of fusion protein)

XpressTM Epitope(detection of fusion protein)

EK site(enterokinase 에 의해 잘리는 site)

attR1,attR2(recombination site)

CmR, ccdB(selective marker)

NdeI, NheI (6x His를 선택할 수 있음)


pEXP1-DEST SystemTM


pEXP2-DEST SystemTM

Gene of interest 의 C-terminal 에

epitope와 purification seq. 를 붙임

size 4.4 kb

pUC origin(high-copy number)

Ampicillin resistance gene

ZeocinTM(selection marker in yeast)

Stop codon

T7 promoter

attR1,attR2(recombination site)

CmR, ccdB(selective marker)

V5 epitope(detection of fusion protein)

6xHis (rapid purification of fusion protein)

AgeI, PmeI (6x His를 선택할 수 있음)


pEXP2-DEST SystemTM


Problems which may arise using in vitro System System

*The synthesis of large proteins may not be very efficient

*Polypeptides which remain unfinished after incubation

*Artefact are fairly common in vitro

*Not all proteins contain methionine[35S]

*empirical


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