The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris |
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Authors: | Geoff P Lin-Cereghino Carolyn M Stark Daniel Kim Jennifer Chang Nadia Shaheen Hansel Poerwanto Kimiko Agari Pachai Moua Lauren K Low Namphuong Tran Amy D Huang Maria Nattestad Kristin T Oshiro John William Chang Archana Chavan Jerry W Tsai Joan Lin-Cereghino |
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Institution: | 1. Department of Biological Sciences, University of the Pacific Stockton, CA 95211, USA;2. Department of Chemistry, University of the Pacific, Stockton, CA 95211, USA |
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Abstract: | The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. |
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Keywords: | Ala alanine AP alkaline phosphatase BMGY buffered minimal glycerol-complex medium BMMY buffered minimal methanol-complex medium CalB Candida antarctica lipase B DMSO dimethyl sulfoxide dNTP deoxyribonucleoside triphosphate ER endoplasmic reticulum Glu glutamic acid HCl hydrochloric acid HPLC high-performance liquid chromatography HRP horseradish peroxidase MATα α-mating factor secretion signal P Pichia PCR polymerase chain reaction TBE tris borate ethylenediaminetetraacetic acid TMB 3 3&prime 5 5&prime -tetramethylbenzidine YNB yeast nitrogen base YPD yeast peptone dextrose |
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