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查看更多产品信息 PichiaPink™ Secretion Signal Set - FAQs (A11155)
67 个常见问题解答
SMD 1168 Pep4 gene disrupted
SMD 1165 PrB gene disrupted
SMD 1163 Pep4/PrB gene disrupted
PichiaPink Strain 2 Pep4 gene disrupted
PichiaPink Strain 3 Prb1 gene disrupted
PichiaPink Strain 4 Prb1, Pep4 gene disrupted
The Pep4 deficient mutant theoretically reduces the protease activity of Proteinase A, Caboxypeptisae Y, and approximately one-half of Proteinase B activity. The proteinase B deficient strain only reduces the activity of proteinase B. Finally, the Pep4/PrB strain reduces or eliminates the proteolytic activity of all three of these enzymes, proteinase A, Carboxypeptidase Y and Proteinase B. These protease deficient strains when compared to wild-type Pichia strains have shown to be highly efficient expression systems for the production of proteolytically sensitive products.
The PRB1 deficient mutant is deficient in expression of proteinase B.
PREPARATION OF PROTEASE DEFICIENT STRAINS
The preferred method for preparing Pichia strains deficient in proteolytic activity, specific disruption of protease-encoding genes, was achieved by gene addition, gene replacement or a combination of additions and replacement referred to as "pop-in-pop-out" method. In gene replacement, the endogenous target gene is physically removed from the target locus, and replaced with a modified gene. This a accomplished by transforming the host with a linear fragment having ends which are homologous to the 5' and 3' ends of the target gene respectively. Gene addition involves adding the transforming DNA to the endogenous target gene. Depending on the manner in which the modified gene of the transforming DNA was altered, gene addition can result in the presence of either two non-functional copies of the target gene, or one functional and one non-functional copy of the target gene. Each of the two copies consists of a portion of the transforming DNA. If a functional copy of the target gene remains after gene addition, it can be removed by homologous recombination between the two copies of the target gene. The combination process of gene addition followed by homologous recombination constitutes the "Pop-in-pop-out" process.