1.Expression Of Bovine Protamine In Pichia Pastoris 2.Cloning Of Mutant Bovine Myostatin Active Domain Coding Sequence And Establishment Of Its Eukaryotic Expression Vector

Pichia pastoris is hererologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. It has many of the advantages such as protein processing, protein folding, and posttranslational modification, while being easy to manipulate and giving higher expression levels. Bovine protamine genomic gene was cloned by PCR amplification and was cloned into pPIC9K, a vector allows secretion of desired protein into the medium, to construct vector p9K-300. Optimized-code bull protamine cDNA gene was cloned by synthesis-PCR and was cloned into pPIC9K vector to construct vector p9K-160.Linearize the vector p9K-300, p9K-160 and pPIC9K with SalI to transform pichia pastoris strain GSl 15 by eletroporation. Positive transformants were selected on the MD plates without histidine. Screening these transformants by G418 we can characterize them for the copy number of the gene of interest. We also analyze these transformants to determine if the gene of interest has integrated into the Pichia genome by using direct PCR screening. Recombinant strains were cultured and induced to express heterologous proteins, and the concentrated supernatants were analyzed by Urea-SDS-PAGE and silver stain. The result indicated that the wild bovine protamine gene couldn't be expressed while the optimized bull protamine gene could be expressed, but the expression level was low. The expression level has no correlation with the copy number.