Construction Of Non-fusion Expression Vectors

As a result of the backwoods coli system can the high level expression exogenous gene, at present obtained the widespread application in the genetic engineering product research and the production. The backwoods coli system expression's reorganization protein majority to forgive the build to become, this requests in the post-processing craft to forgive the body to carry on complex. The existing each kind of complex technology to forgives body's complex efficiency not to be high, is very difficult to some structure more special reorganization protein to realize complex. In the usual situation, in the backwoods coli cytoplasm assumes restores the environment, cannot form the disulphide group key, then the influence reorganization protein conformation's formation, forms the wrong fold in the spatial structure, causes to forgive body's formation.This research construction expresses the material particle, and inserts the exogenous gene, enables it with sulfur oxygen also protein A(trxA), Japanese blood sucking insect Gu Guanggan the sulfenyl transferase (GST) to constitute many along counter-, copies mRNA, after the translation, the internal oxidation returns to original state micro situation, thus presses hundred extraneous source proteins the disulphide group to form correctly, and by may dissolve the form to express in massively Yu Baozhi.This studied constructs successfully expression vector psYpu-3b, but also confirmed the experimental design material particle structure not to have the anticipated tentative plan stability originally; And constructed expression vector psYpu-3a, has carried on reorganization screening, carried on the sequence determination confirmation, has studied in pGEX-4T-2, psYpu-3the GST protein induction expression and may dissolve the situation, obtained in the screening reorganization process not to be suitable uses colony PCR and plasmid PCR, but after being suitable uses the enzyme cut the analysis and the mycelium induction expression, the SDS-PAGE electrophoresis choice masculine gender clone conclusion; Constructed expressed material particle pSYPU-3a-AGAP, through the 2pSYPU-AGAP soluble confirmation research, verification test successfully has constructed material particle pSYPU-3a, caused TrxA and GST, in T7 started under the control altogether copied and translates two independent peptide chains. GST expresses and altogether expresses with TrxA when the solubility alone is not good, but the TrxA expression's solubility is very good. Had proven the material particle pSYPU-3a-AGAP construction confirmed expression vector pSYPU-3a to be advantageous to the exogenous gene soluble expression.