| Title: | Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae |
| Author(s): | Cook JC; Schultz LD; Huang J; George HA; Herber WK; Ip C; Joyce JG; Mao SS; Markus HZ; Miller WJ; Sardana MK; Lehman ED; |
| Address: | "Department of Virus and Cell Biology, Merck Research Laboratories, West Point, Pennsylvania, 19486, USA" |
| ISSN/ISBN: | 1046-5928 (Print) 1046-5928 (Linking) |
| Abstract: | "A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide. Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium. An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels. Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products. A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium. This scheme employed cation-exchange chromatography and reversed-phase HPLC. Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture" |
| Keywords: | "Amino Acid Sequence Animals Arthropod Proteins Base Sequence Chromatography, High Pressure Liquid Chromatography, Ion Exchange Cloning, Molecular DNA, Recombinant Genetic Vectors Intercellular Signaling Peptides and Proteins Mating Factor Molecular Sequen;" |
| Notes: | "MedlineCook, J C Schultz, L D Huang, J George, H A Herber, W K Ip, C Joyce, J G Mao, S S Markus, H Z Miller, W J Sardana, M K Lehman, E D eng 1998/08/07 Protein Expr Purif. 1998 Aug; 13(3):291-300. doi: 10.1006/prep.1998.0893" |
