期刊
JOURNAL OF BIOTECHNOLOGY
卷 99, 期 1, 页码 51-62出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/S0168-1656(02)00157-8
关键词
Pichia pastoris; structural genomics; heterologous expression; screening; high-throughput; affinity tags
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 1(-1) culture volume or higher. (C) 2002 Elsevier Science B.V. All rights reserved.
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