10.6084/m9.figshare.4207017.v2 Moolchand Sigar Moolchand Sigar Nitu Maity Nitu Maity Saroj Mishra Saroj Mishra Enhancing granulocyte colony-stimulating factor expression in <i>Pichia pastoris</i> through fusion with human serum albumin Taylor & Francis Group 2016 Fermentation human therapeutics proteins scale-up 2016-12-09 14:40:50 Journal contribution https://tandf.figshare.com/articles/journal_contribution/Enhancing_Granulocyte_Colony_Stimulating_Factor_Expression_in_i_Pichia_Pastoris_i_via_Fusion_with_Human_Serum_Albumin/4207017 <p>Protein fusion technology has emerged as one of the important strategies to increase the level of expression and half-life of therapeutic proteins in heterologous expression systems. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor and is clinically used against neutropenia. Enhanced expression and stability of G-CSF were achieved in <i>Pichia pastoris</i> by the way of constructing a fusion protein with human serum albumin (HSA). The strategy involved polymerase chain reaction (PCR) amplification of fragments corresponding to codon-optimized G-CSF and domain 3 of HSA. Overlapping PCR was used to obtain the full-length fused gene (1,184 bp) with a 15-bp linker sequence comprising of 4 Gly and 1 Ser residues. Extracellular expression was carried out downstream of <i>α</i>-factor secretion signal sequence under the control of alcohol oxidase 1 promoter using pPICZ<i>α</i>B. Excreted protein in the range of 110–380 mg L<sup>−1</sup> was observed among the transformants. Effect of aeration and temperature was investigated in one of the transformants (35) overexpressing fusion protein and levels of G-CSF enhanced by 1.8-fold and 2.3-fold, respectively. Assay of biological activity indicated the fusion protein to retain similar cell proliferation activity as the commercial G-CSF preparation.</p>