Expression and characterization of a recombinant human factor x/protein c chimeric protein

• Main Author: Stenberg, Leisa M.
• Format: Others
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/10101

A fusion cDNA encoding the light chain domains and activation peptide of human factor X (fX; a procoagulant) and the serine protease domain of human protein C (PC; an anticoagulant) was constructed on the premise that the recombinant chimeric protein (fX/PC) would function as an anticoagulant: it was hypothesized that the protein would be targeted to the Tenase complex by the light chain of fX and there exhibit the proteolytic function of activated PC (APC). The fusion cDNA was expressed in BHK and HEK 293A cell lines and fX/PC was purified to homogeneity from conditioned medium by a two-step method employing an immunoaffinity resin and hydroxyapatite chromatography. Structural and functional analyses of the purified protein revealed that BHK cells were not efficient in performing the posttranslational modifications necessary to generate an active fX/PC protein. Incomplete removal of both the propeptide (concomitant with hydrolysis at alternate sites nearby) and internal tribasic peptide, spurious cleavages, and inefficient 7-glutamyl carboxylation were observed. These problems were overcome by a combination of site-directed mutagenesis to improve the —2 propeptide cleavage site (by replacing Thr with Arg), the use of a HEK 293A cell line for expression, and minor modification of the purification protocol. In this way, homogeneous and properly post-translationally modified preparations were obtained. The chimeric zymogen, fX(T⁻²R)/PC, could be activated by both RVV-X and Protac to an amidolytically active serine protease, although the extent and rate of activation by both activators was lower than for PC. The Km of Protac-activated fX(T⁻²R)/PC for the substrate Spectrozyme PCa (0.14 mM) was comparable to that of APC. The zymogen form of fX(T⁻²R)/PC had no effect on clotting time in APTT assays, whereas the Protac-activated enzyme extended the clotting time in a dose-dependent manner. However, compared with APC, the anticoagulant activity of the chimeric protein was much reduced, suggesting that it may have functioned merely as a competitor for the binding site of either fX within the Tenase complex or activated fX within the Prothrombinase complex. Thus, it can be concluded that, despite substitution of the light chain and activation peptide of PC with those of fX, the fX(T⁻²R)/PC chimeric protein retained some essential features of PC but did not function as hypothesized. === Medicine, Faculty of === Medical Genetics, Department of === Graduate