Heterologous Protein Secretory Expression From Pichia Pastoris With Large Protein As Pro-region

As the representative of the second generation of yeast expression system, Pichia Pastoris, a methylotrophic yeast, has become a well-known and highly successful eukaryotic expression system. It possesses many advantages that other expression systems don't have and has been applied to both industrial manufactures and basic laboratory researches. Due to Pichia pastoris relatively low levels of native secreted proteins, purification of secreted recombinant proteins is possible simply. Proteins secreted from eukaryotic cells required an N-terminal signal peptide. A signal peptide can still maintain its functionality when it is transposed from the context of one protein to another. Foreign genes may be cloned in Pichia pastoris vector to align them in the correct reading frame with the Saccharomyces cerevisiaeαmating factor prepro-peptide. The Saccharomyces cerevisiaeαmating factor prepro-signal is the most widely used secretion signal in Pichia pastoris. During translocation to the ER, the pre peptide is removed. After export of the pro-protein from the ER, the pro-region is proteolytically processed from the precursor in the trans-Golgi. Cleavage occurs at pairs of dibasic amino-acid residues. The length of the pro-region may vary from a few amino acid to several hundred. The pro-region play a important role in the protein secretion such as act as a molecular chaperone or may function as sorting signals or to promote the correct folding of the protein. When the pro-region are deleted from the genes encoding these proteins, secretion can not happen.Considering the significant role of pro-region in the protein secretion, we used two large protein HSA, MBP as the pro-region to construct secreted expression vectors. The protein IFNα2b was used as a reporter. The expression cassette comprised the pre-peptide ofα-factor, the large protein as pro-region, the Kex2 dibasic endopeptidase cleavage site and the target protein. The gene expression was controlled by the inducible AOX1 promoter, Pichia pastoris as a host. The result show that HSA, MBP both can use as effective pro-region lead IFNα2b secreted into culture medium. In particular, MBP was most effective. The N-terminal sequencing of IFNα2b verified that MBP was correctly removed by in vivo Kex2 protease upon the exit of fusion protein from the Golgi complex. Western-blotting confirmed that MBP retained in the secretory pathway. The present result suggested that MBP could utilized as a pro-region to construct new secretion vector in the expression system designed for the secretory production of other heterologous protein from Pichia pastoris.