Expression of the Full-length Human Recombinant Keratinocyte Growth Factor in Pichia pastoris

Document Type : Research Articles

Authors

• zahra bahadori ¹
• seyed javad Mowla ¹
• Hamid Reza Kalhor ²

¹ Tarbit Modares university

² Sharif University of technology

https://doi.org/10.22067/jcmr.v8i1.50029

Abstract

Keratinocyte Growth Factor (KGF) is a paracrine-acting and epithelium-specific growth factor produced by cells of
mesenchymal origin. Based on preclinical data, recombinant KGF plays a critical role in protecting and repairing of
damaged epithelial tissues. Despite great efforts to express recombinant human KGF(rhKGF) in different organisms,
attempts for finding appropriate protein expression system with the ability of producing a properly folded and
processed KGF needs further investigation. Pichia pastoris has been used successfully and extensively for production
of industrial enzymes and pharmaceutical proteins. Herein, we investigated the affect of pro-region-α-factor early
deletion on production and secretion of rhKGF in Pichia pastoris. Initially, expression of human KGF induced in
MCF-7 cell line treated with 1, 25-Dihydroxy vitamin D3. The coding sequence of full-length rhKGF194 was then
cloned into the yeast integrative expression vector, downstream of α-factor and was integrated into P. pastoris
genome. KGF protein was expressed in P. pastoris x33 cells, usingα-factor signal peptide for translocation of KGF
to ER. An internal human signal peptide was also arranged after α-factorfor early removal of the pro-region in ER.
RT-PCR results demonstrated that KGF mRNA was expressed successfully after induction by methanol.
Recombinant KGF protein expression was detected by Western blotting in cell lysats, but not in conditioned media.
A molecular weight of 17 kD for rhKGF194 indicates that the α-factor and internal human signal peptideshad been
removed in x33 cells. The results indicate that in the absence of pro-region-α-factor, the recombinant KGF protein
was not efficiently processed and transported within the biosynthesis-secretory pathway. As KGF protein is an
unstable growth factor and tend to aggregate because of some native properties, It seems that presence of a chaperon
molecule fusion with KGF is necessary for efficient secretion of the recombinant protein.

Keywords

• Keratinocyte growth factor
• Pichiapastoris
• Signal peptide

20.1001.1.20089147.2016.8.1.1.9

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