High-efficiency Production Of Recombinant Proteins SCF In Pichia Pastoris By Optimizing Kex2Pi’site

In nowdays,the study of the recombinant protein was developing,a variety of proteinexpression systems had been continuously improved. As a classic protein expression system，Pichia pastoris had been extensively studied.Pichia had a high growth rate.The cμltureconditions was simple,less toxic,less pollution.The conformation of secreted protein wascomplete and high activity,while its protein secretion levels instability had become a majorproblem in production and research.For the yeast secretory instability problems,the Pichiapastoris was optimized in Kex2P1’ amino acid site,so that efficiency of Kex2digestion wasgreatly improved. The secretion of the protein was enhanced in Pichia pastoris.To facilitate screening high expression of mutants,the reporter gene Luciferase wasintegrated into the expression vector pPICZαA in research.The Luciferase gene was fused withthe target gene and was expressed together under the appropriate conditions,so that theexpression of the target protein was indirect calibrated by Luciferase reporter gene expression.Luciferase was reacted with specific substrate and released of photons in the presence of O2,ATP and Mg2+.The fluorescence was quantificationally detected by a fluorescent luminometer.Since the Luciferase reporter gene and the target gene are fusion expression，fluorescence valuecould screen high expression of the mutant strain,which was used for follow-up expression andpurification.Then the stem cell factor(SCF) was highly expressed on the basis of optimizing Kex2P1’site.As an important hematopoietic stem cell factor,its functional role was mainly throμghcombination with e-kit receptor,stimμlating mast cells proliferation,coordination with growthfactors to maintain progenitor cells and hematopoietic cell survival.At the same time,SCFplayed an important role on increasing the number of colonies,directed differentiation andadhesion.In this paper, pPICZAα-A as the vector,highly expressed SCF by optimizing the Kex2P1’ site of amino acids.The high activity of SCF was purified by means of steps finally.