Promoter Assessment in Hansenula polymorpha Using a lacZ Reporter Gene
Analysis of the strength and regulatory characteristics of commonly used Hansenula polymorpha-detived promoter elements using LacZ as reporter gene.
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PROTOCOL
Promoter Assessment in Hansenula polymorpha Using a lael Reporter Gene MANF RE DSUCKOW, MARTINA KUTZNER, CARSTEN AMUEL, COR NELI S P. HO LLENBERG, and GERD GELLISSEN
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Aim
Analysis of the strength and regulatory cha racteristics of commonly used Han senula polymo rpha-derived promoter elements using LacZ as reporter gene.
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Introduction
The recognition of th e methylotrophic yeast H. poly morpha as a host for heterologous protein product ion is primarily due to the availability of the two strong promoters FMD (formate dehydrogenase) and MOX (meth anol oxidase) that are derived from methanol metaboli sm genes. Both promoters are well characteri zed and have found successful industrial application in the high level expression of foreign genes (Gellissen and Hollenb erg 1997; Gellissen 2000, 2002).MOX and FMD are co-regulated with respect to carbon source. Repre ssion takes place on glucose, derepression on glycerol, and ind uction on methanol (Gellissen and Hollenb erg 1997). However, high-yield heterologous gene expression is not restricted to condition s of meth ano l induction, but can also be obtained using glycerol derepression or even glucose starvation conditions (Mayer et al. 1999). The FMD proGerd Gellissen (~ ) , Manfred Suckow, Martina Kutzner, Rhein Biotech GmbH, Eichsfelder Str. 11,40595 Dus seldorf, Germany, Tel.: +49-211-75845- 137/157; Fax: +49-211-75845 180; e-mail: [email protected] Cars ten Amuel, Cornelis P. Hollenberg.Jnstltut fUrMik robiologie, Heinrich-Hein e-Universitat, Univers itatast r. 1, 40225 Diisseldorf, Ger many
Springer LabManual K. Wolf. K. Breunig,G. Barth (Eds.) Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology C Springer-Verlag Berlin Heidelberg 2003
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moter, in part icular, was found to elicit excellent expression levels under non-i nduced condit ions. A mor e recent pro moter element is der ived from the TPSl (tre halose-e-phosphate synthase) gene (Reinders et aI.1999). This prom oter was found to elicit a strong constitutive gene expression that can be boosted at elevated temperatu re (Amuel et a1. 2000).To further optimize condit ions for gene expression under control of these promoters, different temperat ure and pH cond itions as well as supplementation with different carbon sources will be assessed on one culture medium (YNB ) using defined recombinant H. poly morpha strains in which production of a report er protein is under control of these promoters. As reporter protein of choice, the lacZ-encoded ~ -gal acto sid as e was selected. The strains were generated by transformation of strain RBll (odel ) with plasmids harbo ring an URA3 gene for complementation of the host's auxotrophy besides th e reporter gene cassette. Transformation of H. polymo rpha typically results in a range of recombin ant strai ns with a variety of copy numb ers fixed for th e individual strains. Since productivity of a strain depends to some extent on the gene dosage, determination of gene copy number is requir ed to exclude
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