Isolation and cloning of the β subunit of human follicle stimulating Hormone (hFSHβ) with its native gene's signal sequence in methylotroph yeast Pichia pastoris pPIC9 shuttle vector

Abstract

Introduction: 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 β 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 α factor signal sequence. Material and Methods: 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. Results: The sequence of FSHβ 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. Conclusion: The new construct, pPIC9F1, contains the coding sequence of FSHβ gene and its signal sequence (E2-IVS2-E3). Therefore, this construct can be used for integration of FSHβ gene into yeast genome exactly downstream to AOX1 promoter. Under this condition, a fusion protein is produced that it contains two signal peptides, α factor and FSH signal peptides. Yeast expression system is able to cleavage α factor. It seems this is the first attempt for cloning of human FSHβ in yeast expression system