Protease activity in protein-free NSO myeloma cell cultures
2005 (English)In: In vitro Cellular & Developmental Biology-Animal, ISSN 1071-2690, E-ISSN 1543-706X, Vol. 41, no 10, 330-336 p.Article in journal (Refereed) Published
Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that thiscell line produced and released/secreted several proteases. Two caseinolytic activities at 45-50 and 90 kDa were identifiedas aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular massesof 30-35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using anenzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid andcysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product oraffecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, butaddition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibodyproduct was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5-4, the aspartic acidproteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM,one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotininsignificantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminalamino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitorand cystatin C.
Place, publisher, year, edition, pages
2005. Vol. 41, no 10, 330-336 p.
conditioned medium, zymography, aprotinin, cathepsin L, SLPI, cystain C
IdentifiersURN: urn:nbn:se:kth:diva-6313DOI: 10.1007/s11626-005-0004-4ISI: 000236070000004PubMedID: 16448222ScopusID: 2-s2.0-33645019941OAI: oai:DiVA.org:kth-6313DiVA: diva2:10993
QC 201009202006-11-012006-11-012014-12-01Bibliographically approved