[Isolation and cloning of the beta subunit of human follicle stimulating hormone [hFSH beta] with its native gene's signal sequence in methylotroph yeast Pichia pastoris pPIC9 shuttle vector]

ABSTRACT

Follicle Stimulating Hormone [FSH] is one of the pituitary glycoproteines that it consists of two subunits; alpha and beta. The beta subunit is responsible for the biological activity of FSH. The aim of present study was isolation of the beta subunit coding sequence containing its signal sequence from human genome and then cloning of the isolated sequence in pPIC9 shuttle vector under the control of AOX1 promoter and ? factor signal sequence. the gene sequence of interest was isolated as a 2kb DNA fragment and cloned in pTZ57R vector resulting to pTV-2019 plasmid. The construct was used as template for modification of 5? region of gene upstream to ATG codon using PCR. Finally, amplicon was cloned in pPIC9 and the new construct named pPIC9F1. The sequence of FSH beta gene in pTV-2019 was confirmed by restriction analysis and DNA sequencing. In addition, restriction analysis and AOX1 primer-mediated PCR showed that pPIC9F1 has correct construction. The new construct, pPIC9F1, contains the coding sequence of FSH beta gene and its signal sequence [E2-IVS2-E3]. Therefore, this construct can be used for integration of FSH beta gene into yeast genome exactly downstream to AOX1 promoter. Under this condition, a fusion protein is produced that it contains two signal peptides, ? factor and FSH signal peptides. Yeast expression system is able to cleavage ? factor. It seems this is the first attempt for cloning of human FSH beta in yeast expression system