β-lactamase reporter system for selecting high-producing yeast clones

        In the production of therapeutic proteins in biopharmaceutical industry, it is needed to develop a good screening and selection method for highly-productive clones to lower the production cost. In yeast Pichia pastoris, it has been shown to be a good protein expression system because a new strain with mammalian-type glycosylation was found. However, in P. pastoris, high expression clones are conventionally selected by using different concentrations of antibiotics to screen strains with multiple copies of the expression vector. This method has its limitation due to the limitation in number of colonies screened and the protein expression level is not always correlated with vector copy numbers. Another way of determining the high expression clones is to use GFP as a reporter protein, but the fusion protein is not a good choice for pharmaceutical proteins.

A rapid and simple method to select highly-productive clones is created. This novel expression vector contains two independent expression cassttes. One of these has multiple cloning sites to insert the gene of interest (tested by GFP as a model) controlled by a strong, inducible promoter, AOX1; the other one is consisted of a β-lactamase reporter gene under the control of a weak promoter, YPT1. Clones producing high level of GFP can be selected directly on the plate depending on the intensity of brown color product by β-lactamase. The activity of β-lactamase was found to be proportional to the fluorescence of GFP, which indicated the production level of GFP protein. This reporter system can further be applied to other yeast expression systems such as S. cerevisiae and H. polymorpha.

Reference:β-lactamase reporter system for selecting high-producing yeast clones. BioTechniques 44:477-484 (April 2008)

Yi-Lin (Irene) Cheng
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