Figure 1.

Engineering novel bifunctional fusions of CD39-CD73 and control fusion proteins. A: novel CD39-CD73 bifunctional chimeric proteins were bioengineered by fusing the ectodomain (ECD) of human CD39 and CD73 to either the NH₂- or COOH-terminus of the Fc region of human IgG₁ and to the other domain through gene synthesis and PCR cloning, as described in materials and methods. To generate activity comparison controls, human CD39-ECD-Fc fusion (CD39CH23), human CD73-ECD-Fc fusion (CD73CH23), human alkaline phosphatase ECD-Fc fusion (ALPCH23), and human acid phosphatase ECD-Fc fusion (HAPCH23) were also generated through gene synthesis and PCR cloning, as described in materials and methods. B: production of CD39-CD73 bifunctional fusions and the control fusion proteins. The resulting constructs shown in A were then transfected into Expi293F cells as described in materials and methods. Chimeric recombinant proteins, secreted into conditioned media, were affinity-purified through protein A resin and buffer-exchanged, as described in materials and methods. As described, 4 µg of protein samples was run, under reduced conditions, on 4%–12% SDS-PAGE and then stained with Coomassie blue. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.