The catalytic subunit (L-microCANP) of human
calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-mu CANPDelta3) and the
calmodulin-like domain IV (L-mu CANPDelta4), respectively. The bacterially expressed
proteins were solubilized from the inclusion bodies and refolded with
polyethylene glycol. In Sf9 cells, co-expression of the inhibitor
calpastatin was necessary to prevent
autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of
mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively. Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its
autolysis. A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of
calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells. This mutant required less Ca2+ (about 50 microM) than native erythrocyte
calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations. All
recombinant proteins were active as monomers in
polyethylene glycol-containing
buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed
proteins. Activation by Ca2+ promoted the separation of the two subunits of the expressed
recombinant proteins.