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Producing functional recombinant human keratinocyte growth factor in Pichia pastoris and investigating its protective role against irradiation

Bahadori, Z ; Sharif University of Technology | 2018

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  1. Type of Document: Article
  2. DOI: 10.1016/j.enzmictec.2018.01.003
  3. Publisher: Elsevier Inc , 2018
  4. Abstract:
  5. Keratinocyte Growth Factor (KGF) is a paracrine-acting, epithelial mitogen that plays a prominent role in the regeneration of damaged epithelial tissues. In spite of different attempts to produce recombinant human KGF in many organisms, including bacteria, mammalian cells, plant cells and insect cells; production of recombinant form suffers from lower yields and recovery relative to other recombinant proteins of similar size and properties. Due to many advantages of Pichia pastoris expression systems for producing industrial enzymes and pharmaceutical proteins, in this study P. pastoris was chosen as a host for KGF expression. For preparing human KGF coding sequence, MCF-7 cell line was treated with 1,25-Dihydroxy vitamin D3 for inducing the expression of KGF. The coding sequence of 23N-terminal truncated KGF form was amplified using RT-PCR technique and then cloned into the yeast expression vector in frame with the yeast α-factor secretion signal. The recombinant plasmid was integrated into Pichia pastoris strain X-33 genome. Western blotting and Mass Spectrometry demonstrated that recombinant human KGF (rhKGF) was correctly expressed after methanol induction and secreted into the media. The recombinant protein was purified from the media by heparin affinity chromatography. MTT assay showed that the purified rhKGF had a proliferative effect on NIH3T3 and A549 cell lines. In addition, protective effect of recombinant KGF was assessed in A549 cell line after irradiation. The results showed that the recombinant protein was biologically active. Finally, the effect of recombinant KGF was investigated on proliferation of MCF-7 cell line and its response to radiation. The results showed that pre-treatment of KGF have a protective effect on MCF-7 cell line after irradiation. © 2018
  6. Keywords:
  7. Cytoprotection ; KGF ; Recombinant protein ; Cell culture ; Cells ; Cloning ; Mammals ; Molecular biology ; Polymerase chain reaction ; Purification ; Tissue regeneration ; Yeast ; Heparin affinity chromatography ; Human keratinocyte ; Keratinocyte growth factors ; Pharmaceutical protein ; Pichia Pastoris ; Proliferative effects ; Recombinant plasmid ; Calcitriol ; Heparin ; Methanol ; Palifermin ; FGF7 protein, human ; Keratinocyte growth factor ; Radioprotective agent ; A-549 cell line ; Affinity chromatography ; Amino terminal sequence ; Article ; Cell protection ; Controlled study ; Drug mechanism ; Expression vector ; Fungal genome ; Fungal strain ; Gene expression system ; Human ; Human cell ; Irradiation ; Komagataella pastoris ; Mass spectrometry ; MCF-7 cell line ; Molecular cloning ; MTT assay ; NIH 3T3 cell line ; Nonhuman ; Protein expression ; Protein purification ; Protein synthesis ; Radiation dose ; Radiation protection ; Reverse transcription polymerase chain reaction ; Western blotting ; Animal ; Biosynthesis ; Drug effect ; Genetics ; Metabolism ; Mouse ; Pichia ; A549 Cells ; Animals ; Cell Proliferation ; Cloning, Molecular ; Fibroblast Growth Factor 7 ; Humans ; MCF-7 Cells ; Mice ; NIH 3T3 Cells ; Pichia ; Radiation tolerance ; Radiation-Protective agents ; Recombinant Proteins
  8. Source: Enzyme and Microbial Technology ; Volume 111 , April , 2018 , Pages 12-20 ; 01410229 (ISSN)
  9. URL: https://www.sciencedirect.com/science/article/pii/S0141022918300097