Michele L. Rollence

Engineering a Stable Protease

We have used several approaches to engineer large increases in the stability of the Bacillus serine protease, subtilisin. These include introducing disulfide cross-links, improving electrostatic interactions at calcium ion binding sites, and the use of in vitro random mutagenesis coupled with a phenotypic screen to identify stabilizing mutational events. More than twenty individual stabilizing mutations of subtilisin BPN’ have been identified. Thermodynamic analysis has shown that individually these modifications contribute between 0.3–1.5 Kcal/mol to the free energy of stabilization. We have further found that combining individual stabilizing mutations results in cumulative increases in stability. Calorimetric and crystallographic data demonstrate that increases in the free energy of stabilization are often independent and additive. We therefore have been able to create extremely stable versions of subtilisins in a step by step manner. Thermodynamic stability of subtilisin was also shown to be related to resistance to irreversible inactivation at high temperature and high pH. The most stable versions have half-lives at high pH or high temperature approaching 1000-times longer than the wild type subtilisin BPN’.

Production of engineered IgM-binding single-chain antibodies inEscherichia coli

SummaryTwo single-chain antibodies were engineered and tested as novel binding proteins with specificity for immunoglobulin M. Genes for the two single-chain Fv proteins were assembled from the variable light chain cDNA and variable heavy chain cDNA of monoclonal antibodies DA4.4 and Bet 2, which specifically bind human IgM and mouse IgM, respectively. Both single-chain Fv proteins were designed with a 14-amino acid linker which bridged the variable light chain and variable heavy chain domains. The two proteins were expressed inEscherichia coli, purified and assayed for IgM-binding activity. Both proteins demonstrate a binding specificity for their corresponding IgM which is similar to the monoclonal antibodies from which they were derived. These small IgM-binding proteins may have applications in the investigation of the immune response and in the detection and purification of monoclonal antibodies, cell-associated antibodies, and IgM from serum.