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  • Protease-Deficient Strains

    Several protease-deficient strains – SMD1163 (his4 pep4 prb1), SMD1165 (his4 prb1), and SMD1168 (his4 pep4) – have been shown to be effective in reducing the degradation of some foreign proteins (White et al. 1995; Brierley 1998). This is especially noticeable in fermenter cultures of secreted recombinant proteins, because the combination of high cell density and lysis of a small percentage of cells results in a relatively high concentration of these vacuolar proteases. Unfortunately, these protease deficient cells are not as vigorous as wild-type strains with respect to PEP4. In addition to lower viability, they possess a slower growth rate, are more difficult to transform, and are more difficult to handle in shake flask and fermenter cultures. Therefore, the use of protease-deficient strains is only recommended in situations where other measures to reduce proteolysis (e.g., addition of carrier compounds such as case amino acids and/or peptone to the culture medium) have yielded unsatisfactory results. In particular, it is recommended that a wild-type P. pastoris strain and not a protease-deficient strain be utilized as the initial strain for expression studies.

    An additional protease-deficient strain, SMD1168 (Δpep4::URA3 Δkex1::SUC2 his4 ura3), was developed to inhibit proteolysis of murine and human endostatin (Boehm et al. 1999). The Kex1 protease cleaves carboxy-terminal lysines and arginines. Therefore, the KEX1-deleted strain was generated to inhibit carboxy-terminal proteolysis and proved to substantially reduce this type of degradation.

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