Journal
PROCESS BIOCHEMISTRY
Volume 115, Issue -, Pages 146-151Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2022.02.008
Keywords
C4ST-1; Trigger factor; CSA; Recombinant protein; Enzymatic synthesis
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This study established a method for high expression of recombinant C4ST-1 in Escherichia coli, enabling the production of non-animal-derived CSA.
Chondroitin 4-O-sulfotransferase-1 (C4ST-1) is a key enzyme for the biosynthesis of chondroitin sulfate A (CSA). Several past reports have evaluated the expression of C4ST-1 in bacteria. Since N-glycosylation is critical for the activity of C4ST-1, its activity was quite low. Here, we established a method for inducing a high expression of recombinant C4ST-1 in Escherichia coli (E. coli) by using a trigger factor (TF) fusion system. This approach enabled the expression of 18.9 +/- 5.0 mg/L of TF-fused C4ST-1 (TF-C4ST-1). Although TF-C4ST-1 does not undergo N-glycosylation, the k(cat)/K-m was approximately 60% of that of N-glycosylated C4ST-1. By using TF-C4ST-1 and chondroitin (CH) obtained from the culture of another recombinant E. coli, we succeeded in the 10-mg-scale preparation of CSA consisting almost entirely of the CSA disaccharide, GlcA beta 1-3GalNAc(4S). These results demonstrated that the expression of C4ST-1 in bacteria is sufficient to achieve the long-term goal of manufacturing non-animal-derived CSA.
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