Engineering a Stable Protease

Bryan, Philip N.; Rollence, Michele L.; Wood, Jay F.; Quill, Steven; Dodd, Steven W.; Whitlow, Mark; Hardman, Karl D.; Pantoliano, Michael W.

doi:10.1007/978-94-009-1371-4_6

Cited by 5 publications

(5 citation statements)

References 21 publications

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“…It thus appears that the intended disulfide bond was formed spontaneously either as the protein was synthesized or as the protein was secreted into the fermentation broth. These observations are consistent with results previously reported for five other disulfide-containing variants of subtilisin BPN' (Bryan et al, 1985;Pantoliano et al, 1985).…”

Section: Resultssupporting

confidence: 93%

“…0006-2960/87/0426-2077S01.50/0 Previous efforts to introduce intrachain disulfide bonds into proteins have utilized proteins that already contain free cysteine residues, making the choice of where to introduce a second cysteine mate relatively straightforward (Perry & Wetzel, 1984;Villafranca et al, 1983). An early attempt to introduce disulfide bonds ab initio into a protein containing no cysteine residues was made by Bryan et al (1985), using the cloned gene for the bacterial protease subtilisin BPN'. Although none of these initial disulfide-containing subtilisin variants exhibited enhanced stability, these studies provided the first evidence that engineered disulfide bonds will form in vivo in biological systems where the engineered protein can be secreted into the growth medium.…”

mentioning

confidence: 99%

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Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond

Pantoliano¹,

Ladner²,

Bryan³

et al. 1987

Biochemistry

222

117

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Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers.

show abstract

Section: Resultssupporting

confidence: 93%

mentioning

confidence: 99%

Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond

Pantoliano¹,

Ladner²,

Bryan³

et al. 1987

Biochemistry

222

117

View full text Add to dashboard Cite

show abstract

“…As a result, purely random evolution of a biocatalyst is especially useful to improve more global (cumulative) properties, such as stability, rather than the native activity or specificity of an enzyme, which usually depends on synergistic mutational events. Recent studies on the improvement of protease stability came, for example, from Sattler et al and added to a list of earlier studies in this field . The authors randomly mutated subtilisin and successfully screened for enhanced thermostable variants by temperature-gradient gel electrophoresis.…”

Section: Genetic Enzyme Modificationsmentioning

confidence: 99%

Proteases in Organic Synthesis

2002

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“…Most of the effort to increase the thermal stability of proteins by site-directed mutagenesis has focused on the introduction of nonnative disulfide cross-links. This has proven successful in some instances but not others (Perry & Wetzel, 1984;Sauer et al, 1986;Pantoliano et al, 1987;Villafranca et al, 1983Villafranca et al, , 1987; Wells & Powers, 1986;Bryan et al, 1985). Analysis of the results is complicated because of the different ways in which stability is measured.…”

Section: Protein Stabilizationmentioning

confidence: 99%