Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris

Background: Human alpha 1-antitrypsin (AAT) is a potent inhibitor of multiple serine proteases, and protects tissues against their harmful effects. Individuals with reduced or abnormal production of this inhibitor need intravenous administration of exogenous protein. In this study, we employed the m...

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Autores principales:	Arjmand,Sareh, Lotfi,Abbas Sahebghadam, Shamsara,Mehdi, Mowla,Seyed Javad
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2013
Materias:	human alpha 1-antitrypsin Pichia pastoris recombinant proteins
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000100004
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spelling	oai:scielo:S0717-345820130001000042013-01-21Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastorisArjmand,SarehLotfi,Abbas SahebghadamShamsara,MehdiMowla,Seyed Javad human alpha 1-antitrypsin Pichia pastoris recombinant proteins Background: Human alpha 1-antitrypsin (AAT) is a potent inhibitor of multiple serine proteases, and protects tissues against their harmful effects. Individuals with reduced or abnormal production of this inhibitor need intravenous administration of exogenous protein. In this study, we employed the methylotrophic (methanol utilizing) yeast Pichia pastoris (P. pastoris) as a preferential host for efficient production and secretion of recombinant AAT. Furthermore, we examined different strategies to maximize the yield of the secreted protein. Results: Our findings revealed that optimizing the codon usage of AAT gene for P. pastoris had positive effects on the level of secreted AAT under the control of inducible alcohol oxidase 1 (AOX1) and constitutive glycerol aldehyde phosphate dehydrogenase (GAP) promoters. Compared to AOX1, the GAP promoter increased the yield of AAT by more than two fold. It was also demonstrated that the human AAT native signal sequence was more effective than the well-known yeast signal sequence, alpha mating factor (α-MF). Doubling gene dosage nearly doubled the production of AAT, though dosages exceeding this limit had negative effects on the yield. Conclusion: P. pastoris is shown to be an efficient expression system for production of recombinant and biologically active AAT. Also different strategies could be used to elevate the amount of this secretable protein.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.16 n.1 20132013-01-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000100004en10.2225/vol16-issue1-fulltext-4
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	human alpha 1-antitrypsin Pichia pastoris recombinant proteins
spellingShingle	human alpha 1-antitrypsin Pichia pastoris recombinant proteins Arjmand,Sareh Lotfi,Abbas Sahebghadam Shamsara,Mehdi Mowla,Seyed Javad Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris
description	Background: Human alpha 1-antitrypsin (AAT) is a potent inhibitor of multiple serine proteases, and protects tissues against their harmful effects. Individuals with reduced or abnormal production of this inhibitor need intravenous administration of exogenous protein. In this study, we employed the methylotrophic (methanol utilizing) yeast Pichia pastoris (P. pastoris) as a preferential host for efficient production and secretion of recombinant AAT. Furthermore, we examined different strategies to maximize the yield of the secreted protein. Results: Our findings revealed that optimizing the codon usage of AAT gene for P. pastoris had positive effects on the level of secreted AAT under the control of inducible alcohol oxidase 1 (AOX1) and constitutive glycerol aldehyde phosphate dehydrogenase (GAP) promoters. Compared to AOX1, the GAP promoter increased the yield of AAT by more than two fold. It was also demonstrated that the human AAT native signal sequence was more effective than the well-known yeast signal sequence, alpha mating factor (α-MF). Doubling gene dosage nearly doubled the production of AAT, though dosages exceeding this limit had negative effects on the yield. Conclusion: P. pastoris is shown to be an efficient expression system for production of recombinant and biologically active AAT. Also different strategies could be used to elevate the amount of this secretable protein.
author	Arjmand,Sareh Lotfi,Abbas Sahebghadam Shamsara,Mehdi Mowla,Seyed Javad
author_facet	Arjmand,Sareh Lotfi,Abbas Sahebghadam Shamsara,Mehdi Mowla,Seyed Javad
author_sort	Arjmand,Sareh
title	Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris
title_short	Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris
title_full	Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris
title_fullStr	Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris
title_full_unstemmed	Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris
title_sort	elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in pichia pastoris
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2013
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000100004
work_keys_str_mv	AT arjmandsareh elevatingtheexpressionlevelofbiologicallyactiverecombinanthumanalpha1antitrypsininpichiapastoris AT lotfiabbassahebghadam elevatingtheexpressionlevelofbiologicallyactiverecombinanthumanalpha1antitrypsininpichiapastoris AT shamsaramehdi elevatingtheexpressionlevelofbiologicallyactiverecombinanthumanalpha1antitrypsininpichiapastoris AT mowlaseyedjavad elevatingtheexpressionlevelofbiologicallyactiverecombinanthumanalpha1antitrypsininpichiapastoris
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Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris

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