Traditional E. coli expression systemthrough constantly optimizationimprove, havea series of advantages, such as shortproduction cycle, high efficiency, low cost, but also exists the defects,cannot complete eukaryotic protein folding and modification, people use thesystem still cannot solve membrane protein and some toxic protein expression,severely restricts the application of e. coli expression system.
To make up for the inadequacy ofprokaryotic expression system, people commonly used eukaryotic expressionsystem to express recombinant protein, but the eukaryotic expression system havesteps trival, long cycle, low protein production and high costproblems. Also seta higher threshold for eukaryotic expression system application.At present,along with the deepening research for protein expression system, the cell freeprotein expression is from maybe become a reality, which solved the membraneprotein and toxicity protein not express in E. coli expression system or onlyexisted in inclusions problem.
E. coli cell free expression system iscurrently the most common E. coli S30 extract expression system.E. coli S30Extract system use omp T intracellular proteinase and lack of lon proteaseactivity of the mutant strains of E. coli B to prepare, by adding substancessuch as amino acids, T7 RNA polymerase and energy to realize the purpose ofprotein expressed.
E. coli cell free expression system preparationpurpose protein basic principle are as follows: 1, plasmid DNA, or direct PCRproducts, under RNA polymerase synthesis mRNA in vitro;2, the use of cells inthe extract of transcription factors, various kinds of synthetic protein enzymeand plus a supplement for amino acids, energy material, tRNA translate mRNAinto proteins;3, after translate, the release of mRNA will recycle cell free systemto synthesis protein, mRNA inactivation for repeated several times.
E. coli cell free expression system preparethe target proteinprocess is roughly as follows: 1, E. coli B bacteria liquid preparation,collecting bacteria at logarithmic growth phase;2, E. coli cells broken(ultrasonic, glass beads grinding, high pressure at low temperature, etc.), thepreparation of E. coli S30 extract system;3, Add the amino acid, T7 RNApolymerase and energy substances form reaction system;4, Expression plasmidjoin, incubation, and detection;5, Purpose protein purification.
E. coli cell free expression systemcompared to E. coli expression system, have more advantages:
1, Can directly use PCR gene segment as atemplate express purposeprotein, omit carrier construction, transfection,positive clone selection, expression, such a large number of steps, save a lotof time;
2, can directly implement multiple protein expressedinone systerm at the same time, used for protein interactions to validateexperiments;
3, Through 96 T plate, can implementmultiple proteins expression at the same time, quickly screeningprotein;
4, Marker protein preparation, as well as importspecial amino acid;
5, Implementation transmembrane proteinsexpression, solve some problem of prokaryotic expressed as inclusions;
6, can directlyintervene proteinexpression, to achieve the manipulation of protein expression.
Wuhan EIAab always commit to research and improveproteinexpression system, is coming outa series of protein products, based on E. coliwithout cell expression system, rabbit reticulocyte no cell expression system,wheat endosperm cell free protein expression system, and also accept proteincustom services.
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