Increased expression and secretion of recombinant hINFγ through amino acid starvation-induced selective pressure on the adjacent HIS4 gene in Pichia pastoris

Razaghi, Ali, Huerlimann, Roger, Owens, Leigh, and Heimann, Kirsten (2015) Increased expression and secretion of recombinant hINFγ through amino acid starvation-induced selective pressure on the adjacent HIS4 gene in Pichia pastoris. Acta Facultatis Pharmaceuticae Universitatis Comenianae, 62 (2). pp. 1-8.

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Abstract

Transcriptional co-regulation of adjacent genes has been observed for prokaryotic and eukaryotic organisms, alike. High levels of gene adjacency were also found in a wide variety of yeast species with a high frequency of co-regulated gene sets. The aim of this research was to study how selective pressure on the Histidinol dehydrogenase gene (HIS4), using amino acid starvation, affects the level of expression and secretion of the adjacent human interferon gamma gene (hIFNγ) in the recombinant Pichia pastoris GS115 strain, a histidine-deficient mutant. hIFNγ was cloned into the pPIC9 vector adjacent to the HIS4 gene, a gene essential for histidine biosynthesis, which was then transformed into P. pastoris. The transformed P. pastoris was cultured under continuous amino acid starvation in amino acid-free minimal medium for ten days, with five inoculations into unspent medium every second day. Under these conditions, only successfully transformed cells (hIFNγ –HIS4+) are able to synthesise histidine and therefore thrive. As shown by ELISA, amino acid starvation-induced selective pressure on HIS4 improved expression and secretion of the adjacent hIFNγ by 55% compared to unchallenged cells. RT-qPCR showed that there was also a positive correlation between duration of amino acid starvation and increased levels of the hIFNγ RNA transcripts. According to these results, it is suggested that these adjacent genes (hIFNγ and HIS4) in the transformed P. pastoris are transcriptionally co-regulated and their expression is synchronised. To the best of the knowledge of the authors; this is the first study demonstrating that amino acid starvation-induced selective pressure on HIS4 can alter the regulation pattern of adjacent genes in P. pastoris.

Item ID: 42240
Item Type: Article (Refereed Research - C1)
Keywords: human interferon gamma; histidinol dehydrogenase; Gcn4p; serial passage; transcriptional co-regulation
Additional Information:

© by Kirsten Heimann. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. (CC BY-NC-ND 4.0)

ISSN: 1138-6786
Funders: Advanced Manufacturing Co-operative Research Centre (AMCRC)
Projects and Grants: AMCRC 2.3.4
Date Deposited: 23 Feb 2016 00:22
FoR Codes: 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060407 Genome Structure and Regulation @ 40%
10 TECHNOLOGY > 1003 Industrial Biotechnology > 100306 Industrial Molecular Engineering of Nucleic Acids and Proteins @ 40%
07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070401 Aquaculture @ 20%
SEO Codes: 86 MANUFACTURING > 8608 Human Pharmaceutical Products > 860801 Human Biological Preventatives (e.g. Vaccines) @ 50%
83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830199 Fisheries - Aquaculture not elsewhere classified @ 30%
82 PLANT PRODUCTION AND PLANT PRIMARY PRODUCTS > 8299 Other Plant Production and Plant Primary Products > 829999 Plant Production and Plant Primary Products not elsewhere classified @ 20%
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