Rational design of a tripartite fusion protein of heparinase I enables one-step affinity purification and real-time activity detection

► Tripartite fusion proteins of heparinase I were constructed and compared. ► Effects of fusion order and linker sequence on characteristics of fusion proteins were studied. ► Linker sequences have significant effects on protein expression, heparinase I activity and thermostability. ► Optimization of linker combination at different domain junctions were studied.