Isolation and characterization of human tissue kallikrein produced in Escherichia coli: biochemical comparison to the enzymatically inactive prokallikrein and methionyl kallikrein.
This report describes bacterial expression, isolation, and characterization of human tissue kallikrein recombinantly produced in Escherichia coli. Successful production of enzymatically active recombinant human kallikrein requires the following processes: expression, solubilization and refolding of prokallikrein, thermolysin activation, and chromatographic separation. All experimental data confirmed that bacterially derived human kallikrein is properly folded and exhibits expected biochemical functions. As confirmed by SDS-PAGE and reverse-phase HPLC, recombinant kallikrein is apparently pure and is devoid of reduced or other partially folded kallikrein forms. Recombinant kallikrein behaves as a monomeric molecule in solution and exhibits full enzymatic activity in hydrolyzing peptide substrates. The molecule can bind to aprotinin to form kallikrein-inhibitor complex at a 1:1 molar ratio. Peptide mapping analysis derived from pepsin digestion of recombinant kallikrein assigned five disulfide bonds which match those of porcine kallikrein predicted from X-ray structure. Peptides containing unpaired cysteines or mispaired disulfide bonds were not detected. Both properly folded prokallikrein and methionyl kallikrein, containing a propeptide and 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.
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Amgen Center, Amgen Inc., Thousand Oaks, California, 91320, USA.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007610:Kallikreins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013820:Thermolysin / Q000378:metabolism; D020840:Tissue Kallikreins
D004220:Disulfides; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D008715:Methionine; D007610:Kallikreins; C052910:prokallikrein, tissue; D020840:Tissue Kallikreins; D013820:Thermolysin