Beta-lactamase reporter system for selecting high-producing yeast clones.
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.
Hribar
Gorazd
G
Department of Biosynthesis and Biotransformation, National Institute of Chemistry, Ljubljana, Slovenia. gorazd.hribar@ki.si
Smilović
Vanja
V
Zupan
Ana Lenassi
AL
Gaberc-Porekar
Vladka
V
D002999:Clone Cells; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012641:Selection, Genetic; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D049452:Green Fluorescent Proteins; D001618:beta-Lactamases