β-lactamase reporter system for selecting high-producing yeast clones
β-lactamase reporter system for selecting high-producing yeast clones
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DOI：
10.2144/000112730
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发表时间：
2008-04-01
期刊：
影响因子：
2.7
通讯作者：
Gaberc-Porekar, Vladka
中科院分区：
文献类型：
--
作者：
Hribar, Gorazd;Smilovic, Vanja;Gaberc-Porekar, Vladka
In modern production of protein biopharmaceuticals, a good screening and selection method of high-producing clones can dramatically, influence the whole production process and lead to lower production costs. We have emoted a rapid, simple, and inexpensive method for selecting high-producing clones in the yeast Pichia pastoris that is based on the P-lactamase reporter system. By integrating the reporter gene and the gene of interest into the same genome locus, it was possible to use beta-lactamase activity as a measure of the expression level of the protein of interest. A novel expression vector with two independent expression cassettes was designed and tested using green fluorescent protein (GFP) as a model. The first cassette contained the GFP gene under the control of a strong, inducible AOX1 promoter; while the second cassette consisted of the beta-lactamase reporter gene under the control of a weak constitutive YPT1 promotor High-producing GFP clones were selected directly on the plates based on the color change after hydrolysis of the beta-lactamase substrate added to the medium. beta-lactamase activity was found to positively, correlate with GFP fluorescence. The reporter system described is widely applicable - it can be easily applied to other, also pharmaceutically relevant proteins and to other Yeast expression systems, such as Saccharomyces cerevisiae and Hansenula polymorpha.
