Studies On Expression Of Recombinant Human Insulin In Pichia Pastoris

Insulin is a peptide hormone composed of 51 amino acid residues and has a molecular weight of 5808 Da.Insulin is composed of two peptide chains referred to as the A chain and B chain.A and B chains are linked together by two disulfide bonds, and an additional disulfide is formed within the A chain.The A chain consists of 21 amino acids and the B chain of 30 amino acids.It is produced in theβcell in the pancreas and released when any of the several stimuli is detected.Insulin is a hormone which has extensive effects on metabolism and other body functions.The actions of insulin on the global human metabolism level include:control of cellular intake of certain substances,most prominently glucose in muscle and adipose tissue, Increase of DNA replication and protein synthesis via control of amino acid uptake.It increased glycogen,fatty acid synthesis,decreased proteolysis,lipolysis and gluconeogenesis.Since Banting and Best discovered the hormone,insulin in 1921,it was not only considered as the main medication for diabetes mellitus and the most popular protein drugs,but also the model molecular of small protein hormone.It played a leading role in many subjects,such as molecular biology,structural biology and signal transduction.There is a great demand for insulin in medical treatment and research.The insulin was derived from the pancreas of animals at first.But it caused severe adverse effects due to its immunogenicity.The E.coli was used to express the recombinant human insulin which needed complicated processing after expression. There were remaining some technical problems in high class animals and plants expression system.The Pichia pastoris eukaryotic expression system was developed to express recombinant human insulin in this thesis.Recombinant human insulin was generated via two strategies.One is A and B chain combination.The A and B chain first were separately created through genetic engineering,and then-combined to human insulin after protease processing and purification.The other way of insulin generation is from proinsulin or proinsulin analog,which digested by trysinase and carbosypeptidase B to get rid of C peptide. We construct the A and B chain series connection expression vector and take advantage of post-translation modification of yeast expression system to express the recombinant human insulin to form the disulfide bond in the cells,and then secret the insulin out of cells.1.Get the insulin single chain expression vector pPICZα-InsA,pPICZα-InsBWe changed the genetic codons of InsA and InsB to Pichia pastoris favorite codons by artificial synthesis.Cloning vector is pBS(+),cloning site is Xho I/EcoR. The primer inserted XhoⅠtarget site,Kex2 in 5' end,terminal codon taa and EcoRⅠtarget site in 3' end.Kex2 is essential to cutαsignal peptide efficiently. Then we got the recombinant plasmid pBS-InsA,pBS-InsB.After XhoⅠ,EcoRⅠdigestion,the Insulin gene was cloned in frame into the pPICZαC vector between the XhoⅠ(5' end) and Eco RⅠ(3' end) restriction sites to generate the plasmid pPICZα-InsA,pPICZα-InsB.2.Construct the plasmid pPICZαC-InsA△PmeI,pPICZαC-InsB△PmeI with the Pme I mutation.PCR was performed for mutation with the special designed primer using pPICZαas the template.The PCR product and the insulin single chain expression vector were digested by DraⅠand HindⅢ,and then undertake ligation and transformation.The mutation plasmid was identified by PmeⅠdigestion.3.Construct the complete insulin expression plasmidThe insulin single chain expression vector and the PmeⅠmutation vector was separately digested by BglⅡ+ ClaⅠand BamHⅠ+ ClaⅠ,and then ligated.The plasmid can be indentified by EcoRⅠdigestion.4.Expression and identification of recombinant human insulin.After confirmation by endonuclease digestation assay and sequencing, recombinant vector was linearized by PmeⅠ,then transformed into the X-33 via electroporation.Transformed yeasts were spreaded on YPD plates containing Zeocin to isolate-resistant clones.After clones were cultured for 72 hours at 28℃,The single clones were picked into culture.Proliferate the clones in culture fluid BMGY,then induced the expression of Insulin with 0.5%methanol everyday for 7 days.Perform ECL detect recombinant human insulin.In conclusion,our studies succeed in constructing the complete insulin series connection expression plasmid and secreting expression of insulin in Pichia pastoris.