Expression of histidine-tagged bovine growth hormone employing the prokaryotic pET system
Journal Title: IOSR Journal of Biotechnology and Biochemistry (IOSR-JBB - Year 2018, Vol 4, Issue 2
Abstract
As the recombinant bovine growth hormone (rbGH) has become crucial in galactopoiesis research, the main goal of this study was the in vitro generation of histidine-tagged rbGH proteins using the prokaryotic pET system. The isolation of rbgh genes was carried out by PCR using specific primers and a cDNA template retrotranscribed from a sample of total RNA extracted from the bovine pituitary tissue. The PCR products were cloned into the expression vectors pET28a and pET22b. The genetic construction in pET28a added six-histidine residues at the N-terminus region of the rbGH molecule. The genes inserted in pET22b allowed the obtaining of rbGH protein without histidine residues and another variant with the six-histidine residues at the C-terminus region. Different E. coli strains were transformed with the three genetic constructions. The strain RosettaTM (DE3) was selected as the final expression system because it exhibited the highest expression levels among others. After purifying the three rbGH variants, densitometric analysis showed more than 95 % purity in all cases. The proteins also showed biological activity in a cell proliferation assay. This study allowed the obtaining of three actives variants of rbGH with a high purity degree by using the prokaryotic pET system and the E. coli strain RosettaTM (DE3). This approach could contribute to the production of rbGH protein for future galactopoiesis investigations.
Authors and Affiliations
Alaín González Pose, Elianet Lorenzo Romero, Dailenis Abella Matos, Mary Karla Méndez Orta, Liliana Basabe Tuero, Ernesto Manuel González Ramos, Anays Álvares Gutiérrez, Raquel Montesino, Oliberto Sánchez, Jorge Roberto Toledo
Keywords
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