Expression of highly active chondroitin 4-O-sulfotransferase-1 in Escherichia coli by a trigger factor fusion protein expression system

• Published in: Process biochemistry (1991) 2022-04, Vol.115, p.146-151
• Main Authors: Takashima, Makoto, Suzuki, Kiyoshi, Mochizuki, Hideo, Uemura, Satoshi, Inokuchi, Jin-ichi, Eguchi, Tadashi
• Format: Article
• Language: English
• Subjects: biosynthesis; C4ST-1; chondroitin sulfate; CSA; Enzymatic synthesis; enzymes; Escherichia coli; glycosylation; Recombinant protein; Trigger factor
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2022.02.008

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 kcat/Km 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, GlcAβ1–3GalNAc(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. [Display omitted] •Trigger factor fused C4ST-1 (TF-C4ST-1) was expressed in Escherichia coli.•Although TF-C4ST-1 has no N-glycans, it possesses sulfotransferase activity.•The kcat/Km of TF-C4ST-1 was approximately 60% of that of N-glycosylated C4ST-1.•Using TF-C4ST-1, 10-mg-scale preparation of non-animal-derived CSA was possible.•TF-C4ST-1 has sufficient function for manufacturing non-animal-derived CSA.