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231223s2008 xx |||||o 00| ||eng c |
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|a 10.2144/000112730
|2 doi
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|a eng
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1 |
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|a Hribar, Gorazd
|e verfasserin
|4 aut
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| 245 |
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|a Beta-lactamase reporter system for selecting high-producing yeast clones
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|c 2008
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|a Text
|b txt
|2 rdacontent
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|a ƒaComputermedien
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|a ƒa Online-Ressource
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|a Date Completed 03.07.2008
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|a Date Revised 19.11.2009
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|a published: Print
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|a Citation Status MEDLINE
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|a 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 pastoris that is based on the beta-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
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|a Journal Article
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|a Technical Report
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|a Green Fluorescent Proteins
|2 NLM
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|a 147336-22-9
|2 NLM
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|a beta-Lactamases
|2 NLM
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| 650 |
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|a EC 3.5.2.6
|2 NLM
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1 |
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|a Smilović, Vanja
|e verfasserin
|4 aut
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1 |
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|a Zupan, Ana Lenassi
|e verfasserin
|4 aut
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| 700 |
1 |
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|a Gaberc-Porekar, Vladka
|e verfasserin
|4 aut
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| 773 |
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|i Enthalten in
|t BioTechniques
|d 1993
|g 44(2008), 4 vom: 01. Apr., Seite 477-8, 480, 482 passim
|w (DE-627)NLM012627046
|x 0736-6205
|7 nnns
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| 773 |
1 |
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|g volume:44
|g year:2008
|g number:4
|g day:01
|g month:04
|g pages:477-8, 480, 482 passim
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|u http://dx.doi.org/10.2144/000112730
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