High-throughput expression in microplate format in Pichia pastoris.
The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. To serve the increasing demand for clones expressing different cDNAs, we developed a cultivation and induction protocol amenable to automation to increase the number of clones screened for protein expression. Therefore cDNAs are cloned for intracellular expression. The resulting fusion proteins carry affinity tags (6*HIS and StrepII, respectively) at the N- and C-terminus for the immunological detection and chromatographic purification of full-length proteins. Expression is controlled by the tightly regulated and highly inducible alcohol oxidase 1 (AOX1) promoter. The screening procedure is based on a culture volume of 2 mL in a 24-well format. Lysis of the cells occurs via chemical lysis without mechanical disruption. Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones that produce heterologous protein with a yield of 2 mg per L culture volume or higher.
Berlin Technical University, Institute for Biotechnology, Germany.
Mewes
M
Böttner
Christine
C
Lang
D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D024101:Mitochondrial Proteins; D010088:Oxidoreductases / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D000099188:Alternative Oxidase
D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011994:Recombinant Proteins; D000099188:Alternative Oxidase