Engineering Chondroitinase‐Free Baculovirus‐Insect Cell Expression System for Efficient Synthesis of Chondroitin Sulphates

Owing to the high expression levels, proper protein folding and post-translational modifications, baculovirus-insect cell expression system is an ideal platform for producing active chondroitin sulfotransferases, which specifically catalyse the synthesis of chondroitin sulphate (CS). However, the CS can be degraded by the endogenous chondroitinase derived from the viral envelope protein ODV-E66, thus requiring elaborate purification, which is impractical for large-scale production. To address this issue, a chondroitinase-free baculovirus was constructed by deleting the odv-e66 gene from bacterial artificial chromosomes (BACs). Through the engineered baculovirus, highly active chondroitin sulfotransferases (CS-4OST, CS-6OST and GalNAc4S-6OST) were successfully expressed and secreted into the culture supernatant. Of note, the culture supernatant can be directly employed for CS synthesis. Using the cultures harbouring the corresponding sulfotransferases, CS-A, CS-C and CS-E were successfully prepared at the gram-scale, thus obviating the need for laborious and costly purification. Collectively, this work overcomes the inherent limitation of CS degradation in baculovirus-insect cell expression system, thereby providing a robust and reliable platform for the expression of chondroitin sulfotransferases.