High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris.
The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. In order to provide proteins for the 'protein structure factory', a structural genomics initiative, we are working on the high-throughput expression of human proteins. 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 alcoholoxidase 1 (AOX1) promoter. We have developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression. The screening procedure is based on a culture volume of 2 ml in a 24-well format. Lysis of the cells occurs via a chemical lysis without mechanical disruption. Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones which produce heterologous protein with a yield of 5 mg l(-1) culture volume or higher.
Proteinstrukturfabrik, Heubnerweg 6, D-14059 Berlin, Germany.
Mewes
M
Boettner
Bianka
B
Prinz
Caterina
C
Holz
Ulf
U
Stahl
Christine
C
Lang
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D020224:Expressed Sequence Tags; D015200:Fluoroimmunoassay / Q000379:methods; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D000432:Methanol / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D005947:Glucose; D000432:Methanol