Soluble Expression Of Human Proinsulin Analog Gene In Escherichia Coli

In view of the fact that inclusion bodies are formed when Escherichia coli expresses proinsulin makes the subsequent purification cumbersome and complicated and lowers the recovery ratio whereas various unsteady factors exist in the yeast expression system.The work discussed in this thesis using E.coli as an expression system realized the soluble expression of proinsulin E.coli by improving various factors that affect the expression.First of all.a coding framework was designed.adopted the fusion protein expression.introduced his-tag and GBl tag and inserted Lys between the tags and the interest protein so that the tags could be deleted later on.At the same time,the C-peptide was shortened as RRKR to increase the expression quantity and the recombinat efficiency of A、B chains and also the gene order has been optimized to make sure the synthesis of the gene order of human proinsulin analog by means of the codon of E.coli partial analogues without firm RNA necking secondary structure.Later the obtained sequences was inserted into the vectors pET-32a、pNJUTRX-1 and pSYPU-1b kept in the lab to be expressed.The greatest soluble expression quantity was selected and its fermentation conditions were optimized so as to improve expression level.Fermentation conditions were considered mainly in four aspects:temperature.the time of induction, the dosage of IPTG. and inoculation amount to optimize the best expression amount,providing samples for the next step.During purification, first the crude product was obtained by immobilized metal ion affinity chromatography (IMAC) using his tags. After trypsin digestion, again by immobilized metal ion affinity chromatography (IMAC), the component obtained was electrophoretic pure.