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 kallikrein in engineered Chinese hamster ovary cells transfected with a human genomic gene encoding preprokallikrein. At high expression levels, recombinant prokallikrein, an inactive proenzyme form, is predominantly secreted into the culture medium. Upon chromatographic separations, the inactive prokallikrein as well as the mature kallikrein after thermolysin activation of the proenzyme can be prepared to apparent purity. Both prokallikrein and kallikrein can be further separated into two distinct high- and low-molecular-weight isoforms. Kallikrein preparations are fully active in standard kallikrein activity assays such as 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 glycosylated 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 at the detected Asn-linked carbohydrates. In comparison with kallikrein, prokallikrein appears to show a significant decrease in the magnitude of near uv-circular dichroism bands, suggesting a change in local conformation. This conformational change correlates with the loss of activity in proenzyme due to the presence of propeptide.
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Amgen Center, Amgen Inc., Thousand Oaks, California, 91320, USA.
Hsu
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Narhi
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LO
Karkare
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Lin
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FK
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007610:Kallikreins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010449:Peptide Mapping; D011288:Prekallikrein / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D020840:Tissue Kallikreins
D000596:Amino Acids; D004792:Enzyme Precursors; D007527:Isoenzymes; D011994:Recombinant Proteins; D011288:Prekallikrein; D007610:Kallikreins; C051313:preprokallikrein; C052910:prokallikrein, tissue; D020840:Tissue Kallikreins; D013820:Thermolysin