<abstract xmlns="http://www.w3.org/1999/xhtml"><p> 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<sup>+</sup>) 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 starvationinduced selective pressure on HIS4 can alter the regulation pattern of adjacent genes in P. pastoris.</p></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 starvationinduced selective pressure on HIS4 can alter the regulation pattern of adjacent genes in P. pastoris.

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

College of Marine and Environmental Sciences, James Cook University, Townsville 4811, Queensland, Australia

Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Townsville4811, Queensland, Australia

Comparative Genomics Centre, James Cook University, Townsville 4811, Queensland, Australia

College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, QLD, 4811, Australia

European Pharmaceutical Journal

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Transcriptional co-regulation of adjacent genes has been observed for prokaryotic and eukaryotic organisms, alike. High levels of gene adjacency were also...

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

Pubblicato online: 31 dic 2015

Pagine: 43 - 50

Ricevuto: 10 ago 2015

Accettato: 31 ott 2015

DOI: https://doi.org/10.1515/afpuc-2015-0031

Parole chiave
Human interferon gamma, Histidinol dehydrogenase, Gcn4p, Serial passage, Transcriptional co-regulation

© by Kirsten Heimann

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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

Pubblicato online: 31 dic 2015

Pagine: 43 - 50

Ricevuto: 10 ago 2015

Accettato: 31 ott 2015

DOI: https://doi.org/10.1515/afpuc-2015-0031

Parole chiaveHuman interferon gamma, Histidinol dehydrogenase, Gcn4p, Serial passage, Transcriptional co-regulation

© by Kirsten Heimann

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Parole chiave
Human interferon gamma, Histidinol dehydrogenase, Gcn4p, Serial passage, Transcriptional co-regulation