ISOLATION AND CHARACTERIZATION OF HUMAN TISSUE KALLIKREIN PRODUCED INESCHERICHIA-COLI - BIOCHEMICAL-COMPARISON TO THE ENZYMATICALLY INACTIVE PROKALLIKREIN AND METHIONYL KALLIKREIN

This report describes bacterial expression, isolation, and characteriz ation of human tissue kallikrein recombinantly produced in Escherichia coli. Successful production of enzymatically active recombinant human kallikrein requires the following processes: expression, solubilizati on and refolding of prokallikrein, thermolysin activation, and chromat ographic separation. All experimental data confirmed that bacterially derived human kallikrein is properly folded and exhibits expected bioc hemical functions. As confirmed by SDS-PAGE and reverse-phase HPLC, re combinant kallikrein is apparently pure and is devoid of reduced or ot her partially folded kallikrein forms. Recombinant kallikrein behaves as a monomeric molecule in solution and exhibits full enzymatic activi ty in hydrolyzing peptide substrates. The molecule can bind to aprotin in to form kallikrein-inhibitor complex at a 1:1 molar ratio. Peptide mapping analysis derived from pepsin digestion of recombinant kallikre in assigned five disulfide bonds which match those of pot-cine kallikr ein predicted from X-ray structure. Peptides containing unpaired cyste ines or mispaired disulfide bonds were not detected. Both properly fol ded prokallikrein and methionyl kallikrein, containing a propeptide an d an initiator methionine at their N-termini, respectively, were also produced and isolated. These two molecules are structurally similar to recombinant kallikrein, but are not enzymatically active. (C) 1996 Ac ademic Press, Inc.