Production and characterization of human carbonic anhydrase IX in insect cells
Lindfors-Huttunen, Mikaela (2014)
Biokemia - Biochemistry
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Carbonic anhydrase IX (CA IX) is a cell surface enzyme, which catalyzes CO2 hydration. It has a function to maintain favorable pH conditions for cancer cell growth and survival. Expression of CA IX is a marker of hypoxia, which has been noticed to be an indicator for poor prognosis in specific cancers. CA IX has been analyzed intensively during the last decades but some of its biochemical properties were not revealed until this study was conducted. Hence, the recombinant protein forms of CA IX were produced and purified in this study using the baculovirus insect cell expression system. The aim was to produce CA IX recombinant proteins for further studies to analyze the activity and other biochemical properties of these enzymes. The recombinant proteins, which had either the catalytic domain or the proteoglycan domain of CA IX were separately produced, purified and analyzed. Additionally a CA IX form having both domains in the same recombinant protein was also studied. The used baculovirus insect cell expression system was found to be a feasible way to produce recombinant CA IX. It was found that the proteoglycan domain has a notable influence on enzyme activity of CA IX. In addition, activity values confirmed the previously reported results that the catalytic domain of CA IX has average activity as compared to the other CA isozymes. However, the addition of ZnCl2 to the reaction was found to increase the catalytic activity of CA IX, and this property was concluded to be a unique feature for CA IX. In the presence of ZnCl2 the catalytic activity of the full-length recombinant protein having both the catalytic and proteoglycan domains was higher than measured before for any other CAs.
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