Increased soluble heterologous expression of a rat brain 3-O-sulfotransferase 1 - A key enzyme for heparin biosynthesis

Authors
Jin, Weihua
Li, Shuai
Chen, Jiale
Liu, Bing
Li, Jie
Li, Xueliang
Zhang, Fuming
Linhardt, Robert J.
Zhong, Weihong
ORCID
https://orcid.org/0000-0003-2219-5833
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Other Contributors
Issue Date
2018-11-01
Keywords
Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
Degree
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Full Citation
Increased soluble heterologous expression of a rat brain 3-O-sulfotransferase 1 - A key enzyme for heparin biosynthesis, W. Jin, S. Li, J. Chen, B. Liu, J. Li, X. Li, F. Zhang, R.J. Linhardt, W. Zhong, Protein Expression and Purification, 151, 23-29, 2018.
Abstract
Heparan sulfate (HS), is a glycosaminoglycan (GAG) involved in various biological processes, including blood coagulation, wound healing and embryonic development. HS 3-O-sulfotransferases (3-OST), which transfer the sulfo group to the 3-hydroxyl group of certain glucosamine residues, is a key enzyme in the biosynthesis of a number of biologically important HS chains. The 3-OST-1 isoform is one of the 7 known 3-OST isoforms and is important for the biosynthesis of anticoagulant HS chains. In this study, we cloned 3-OST-1 from the rat brain by reverse transcription-polymerase chain reaction (RT-PCR). After codon optimization and removal of the signal peptide, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) to obtain a His tagged-3-OST-1 fusion protein. SDS-PAGE analysis showed that the expressed 3-OST-1 was mainly found in inclusion bodies. The 3-OST-1 was purified by Ni affinity column and refolded by dialysis. The activity of obtained 3-OST-1 was 0.04 U/mL with a specific activity of 0.55 U/mg after renaturation. Furthermore, a co-expressed recombinant plasmid pET-28a-3-OST-1 with the chaperone expression system (pGro7) was constructed and transferred to E. coli BL21 (DE3) to co-express recombinant strain E. coli BL21 (DE3)/pET-28a-3-OST-1 + pGro7. The soluble expression of 3-OST-1 was significantly improved in the co-expressed recombinant strain, with enzyme activity reaching 0.06 U/mL and having a specific activity of 0.83 U/mg. N-sulfo, N-acetylheparosan (NSNAH) was modified by the recombinant expressed 3-OST-1 and the product was confirmed by 1H NMR showing the sulfo group was successfully transferred to NSNAH.
Description
Protein Expression and Purification, 151, 23-29
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Department
The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
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Relationships
The Linhardt Research Labs Online Collection
Rensselaer Polytechnic Institute, Troy, NY
Protein Expression and Purification
https://harc.rpi.edu/
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