Analysis of genetically engineered subtilisin surface microenvironments using high performance liquid chromatography

Roman Michael Chicz, Purdue University

Abstract

Genetically engineered subtilisin variants were obtained for investigation of protein retention in five different chromatographic modes. Earlier work indicated that protein three-dimensional structure sterically defined which surface residues made contact with the stationary phase of chromatographic supports. Using site-directed mutagenesis, variants with multiple surface amino acid substitutions were produced to locate possible contact surface regions. Position 166 was determined to be chromatographically significant, thus single residue substitutions were made to examine the effect, with respect to chromatographic behavior, of the amino acid contributions on the surrounding microenvironment. X-ray crystallography determination, by Genencor, showed no alterations in global protein structure and only slight perturbations at the local level. Cation-exchange retention data was analyzed using the stoichiometric displacement model to quantitate the interactions between the different subtilisins and the stationary phase. Plots of ln k$\sp\prime$ vs. ln 1/ (NaCl), normally yielding a single linear relationship, revealed two lines differing in slope and intercept. The results indicated some salt-induced protein surface event that triggered a conformational change in subtilisin. Selectivity in chromatography can be manipulated through mobile phase pH. Retention time was plotted against mobile-phase pH for cation-exchange (CEC), hydrophobic-interaction (HIC), and immobilized-metal chelate affinity chromatography (IMAC) to ascertain the optimum conditions for variant separations. The sensitivity of CEC was evident by the separation of six subtilisins, differing by only one amino acid out of 275. HIC complemented CEC by resolving variants which coeluted in CEC. Together, the two chromatographic modes were able to separate 10 of the twelve single substitution variants. IMAC was also successful at resolving subtilisin variants. The remaining two modes studied were reversed-phase and hydroxyapatite chromatography. Although subtilisin was strongly retained on both supports, variant separation attempts were unsuccessful. Apparently, the substitutions on neither the multiple nor the single substitution variants were recognized by either mode. This reflects the differences in identification of protein contact surface regions by the various chromatographic methods.

Degree

Ph.D.

Advisors

Regnier, Purdue University.

Subject Area

Biochemistry

COinS