| Autor: |
Hribar, Gorazd, Smilovi, Vanja, Lenassi Zupan, Ana, Gaberc-Porekar, Vladka |
| Zdroj: |
Biotechniques; April 2008, Vol. 44 Issue: 4 p477-484, 8p |
| Abstrakt: |
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 created a rapid, simple, and inexpensive method for selecting high-producing clones in the yeast Pichia pastoristhat is based on the β-lactamase reporter system. By integrating the reporter gene and the gene of interest into the same genome locus, it was possible to use β-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 AOX1promoter, while the second cassette consisted of the β-lactamase reporter gene under the control of a weak constitutive YPT1promotor. High-producing GFP clones were selected directly on the plates based on the color change after hydrolysis of the β-lactamase substrate added to the medium. β-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 cerevisiaeand Hansenula polymorpha. |
| Databáze: |
Supplemental Index |
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