PURIFICATION AND CHARACTERIZATION OF HUMAN TISSUE PROKALLIKREIN AND KALLIKREIN ISOFORMS EXPRESSED IN CHINESE-HAMSTER OVARY CELLS

We report here the expression of recombinant human prokallikrein and k allikrein in engineered Chinese hamster ovary cells transfected with a human genomic gene encoding preprokallikrein. At high expression leve ls, recombinant prokallikrein, an inactive proenzyme form, is predomin antly secreted into the culture medium. Upon chromatographic separatio ns, the inactive prokallikrein as well as the mature kallikrein after thermolysin activation of the proenzyme can be prepared to apparent pu rity. Both prokallikrein and kallikrein can be further separated into two distinct high- and low-molecular-weight isoforms. Kallikrein prepa rations are fully active in standard kallikrein activity assays such a s esterase activity and kinin release from kininogen. Both kallikrein and prokallikrein display multiple molecular forms with differences in both molecular sizes and charges. The structural differences in high- and low-molecular-weight kallikreins or prokallikreins were found to be due to glycosylation, with the high-molecular-weight species glycos ylated at three Asn-linked sites and the low-molecular-weight species at two of the three Asn-linked sites. The multiply charged kallikrein isoforms are derived from different numbers of sialic acids attached a t the detected Asn-linked carbohydrates. In comparison with kallikrein , prokallikrein appears to show a significant decrease in the magnitud e of near uv-circular dichroism bands, suggesting a change in local co nformation. This conformational change correlates with the loss of act ivity in proenzyme due to the presence of propeptide. (C) 1996 Academi c Press, Inc.