Controlling T7 expression system by means of thermal induction

Abstract

The invention discloses a controlling method for constructing a recombination bacterial BL21(G2) for evoked preparation of T7 RNA polymerase by heat, the chromosome of the bacterial includes a T7 gene controlled by lambda P[L] and P[R] double promoters and a cI857 inhibitory gene, the T7 RNA polymerase produced by the recombination bacterial can evoke and activate the T7 promotor, thus manufacturing the destination gene product cloned at the downstream of the promotor.

Description

technical field [0001] The present invention relates to a method for inducing and controlling the gene expression vector (expression vector) in Escherichia coli. More specifically, the present invention is a method for controlling the T7 expression system in a heat-induced manner. This method further includes a segmental formula Feed-fed batch fermentation strategies for high-volume production of recombinant proteins. Background technique [0002] The use of biological cells to produce recombinant proteins (recombinant proteins) with commercial value or medical use is an extremely important and economically forward-looking biotechnology industry. Generally speaking, biological cells that can be used for protein production include microorganisms, insects, animal and plant cells, etc. Among them, the method of using microbial cells to produce recombinant proteins is more economically competitive because microbial cells are easier to culture in large quantities , Fast growth a...

AI Technical Summary

Problems solved by technology

Therefore, the T7 expression system with IPTG as the induction method obviously cannot achieve the purpose of industrial use, and its reason is (1) the price of IPTG is expensive, (2) IPTG is not metabolized by bacterial cells, which easily causes contamination of the fermentation broth and makes the fermentation product Purification is not easy, (3) IPTG is potentially toxic, so it is not suitable for the production of medical products (Figge et al., Cell, 52:713-722, 1988), (4) because IPTG needs the lacY protein of bacterial cells It is transported into cells by active transport, so using a non-saturated amount of IPTG to induce will result in the induced cell population containing induced and non-induced cell populations. This non-uniform state causes the fermented strains to produce different Stability, and it is difficult to achieve the purpose of finely regulating the gene expression of the strain. (5) Due to the insufficient regulation of the lacUV5 promoter, the strain BL21 (DE3) produces a small amount of T7 RNA polymerase under the condition of non-induction, The target gene product that is cloned on the plastid and expressed under the control of the T7 promoter is produced, especially when the target gene product is potentially toxic to the host cell, the growth of the cell will be inhibited, and then the plastid containing the target gene will not be produced. Stable phenomenon (Studier et al., Methods in Enzymology, 185:60-89, 1991), these shortcomings obviously limit the industrial applicability of the T7 expression system

Images