Why are protease deficient Pichia pastoris and Pichia methanolica strains used for protein expression? What strains are available?

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DESCRIPTION OF PROTEOLYTIC ACTIVITIES
Proteinase A is a vacuolar aspartyl protease capable of self-activation, as well as subsequent activation of additional vacuolar proteases, such as carboxypeptidase Y and proteinase B. Carobxypeptidase Y appears to be completely inactive prior to proteinase A-mediated proteolytic processing of the enzyme; proteinase B (encoded by the PrB gene of S. cerevisiae) reportedly is approximately 50% bioactive in its precursor form (i.e. the form that exists prior to proteinase A-mediated processing of the enzyme). Little is known about the proteolytic activities in Pichia pastoris. The following protease deficient Pichia pastoris strains have been made in an attempt to inactivate or delete the homologous proteolytic activities:

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.

USEFULNESS
When proteolytically sensitive recombinant products such as epidermal growth factor (EGF), growth hormone releasing factor (GRF), Insulin-like growth factor-1 (IGF-1), are expressed in Pichia strains which are deficient in proteolytic activity, higher levels of authentic bioactive recombinant product are produced. The following example illustrates the usefulness of these protease deficient strains:

Proteolytic activity of the broth from a normal Pichia strain and a Pep4- Pichia strain were compared by adding known amount of peptide to the broth. The stability of the peptides in the two different broths was monitored by HPLC; when GRF or EFG were incubated with cell-free broth from these two strains, less than 10% of the peptides remained intact after 4 hours. In contrast, the GRF was greater than 60% intact after 4 hours and EGF remained greater than 90% after 8 hours incubation in the cell-free broth of the Pep4- strain. These data demonstrate that the disruption of the pep4 gene of Pichia result in a substantial reduction of the proteolyis.

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