Enhancing granulocyte colony-stimulating factor expression in Pichia pastoris through fusion with human serum albumin.
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 Pichia pastoris 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 α-factor secretion signal sequence under the control of alcohol oxidase 1 promoter using pPICZαB. Excreted protein in the range of 110-380 mg L-1was 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.
a Department of Biochemical Engineering and Biotechnology , Indian Institute of Technology Delhi , New Delhi , India.
Moolchand
M
Sigar
a Department of Biochemical Engineering and Biotechnology , Indian Institute of Technology Delhi , New Delhi , India.
Nitu
N
Maity
a Department of Biochemical Engineering and Biotechnology , Indian Institute of Technology Delhi , New Delhi , India.
Saroj
S
Mishra
D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D005822:Genetic Vectors / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D010843:Pichia / Q000166:cytology / Q000235:genetics* / Q000254:growth & development; D016133:Polymerase Chain Reaction; D000072417:Protein Domains; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012709:Serum Albumin / Q000737:chemistry / Q000235:genetics*; D014170:Transformation, Genetic
D003062:Codon; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D016179:Granulocyte Colony-Stimulating Factor
Fermentation; human therapeutics proteins; scale-up