Cloning, expression and purification of fructosyl amino acid oxidase (FAOX) in Escherichia Coli

Ho Ta Giap (), Phan Ngoc Han, Tran Le Duy Phuong, Phung Thi Thu Phung and Vu Van Van
Additional contact information
Ho Ta Giap: Nguyen Tat Thanh University, Vietnam
Phan Ngoc Han: Nguyen Tat Thanh University, Vietnam
Tran Le Duy Phuong: Nguyen Tat Thanh University, Vietnam
Phung Thi Thu Phung: Nguyen Tat Thanh University, Vietnam
Vu Van Van: Nguyen Tat Thanh University, Vietnam

HO CHI MINH CITY OPEN UNIVERSITY JOURNAL OF SCIENCE - ENGINEERING AND TECHNOLOGY, 2021, vol. 11, issue 1, 20-28

Abstract: Introduction: The level of serum HbA1c is an indicator of the average blood sugar level in the last three months. HbA1c can be quantified using assays involving the enzyme fructosyl amino acid oxidase (FAOX). This study aims to produce GST-tagged FAOX-TE (GST/FAOX-TE), a thermal stable and specific variant of FAOX, for future application studies. Materials and methods: The E. coli strains DH5α and BL21 (DE3) were used as cloning and expression hosts, respectively. The FAOX-TE sequence was synthesized at IDT (US) and clonned into pGEX-4T3 vector, which was confirmed by Colony PCR. The expression was induced at 16°C, 0.5 mM IPTG in LB media containing 50 µg/ml ampicilin. The protein expression profile was analyzed by SDS-PAGE. The cell pellet was sonicated and purified by Glutathione Sepharose 4 Fast Flow (Cytiva, US). The catalytic activity of GST/FAOX-TE with fructosyl valine was determined using high performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD). Results: The fusion protein was successfully expressed in Escherichia coli using the plasmid pGEX-4T3 and purified to high purity 93%. Recombinant GST/FAOX-TE was shown to be active on fructosyl valine. Conclusions: Active GST/FAOX-TE was successfully expressed in E. coli BL21 (DE3) and purified, which will be used for future development of biosensors for fructosyl valine quantification.

Keywords: fructosylamino acid oxidase; FAOX-TE; expression and purification (search for similar items in EconPapers)
Date: 2021
References: View complete reference list from CitEc
Citations:

Downloads: (external link)
https://journalofscience.ou.edu.vn/index.php/tech-en/article/view/1238/1397 (application/pdf)

Related works:
This item may be available elsewhere in EconPapers: Search for items with the same title.

Export reference: BibTeX RIS (EndNote, ProCite, RefMan) HTML/Text

Persistent link: https://EconPapers.repec.org/RePEc:bjw:techen:v:11:y:2021:i:1:p:20-28

DOI: 10.46223/HCMCOUJS.tech.en.11.1.1238.2021

Access Statistics for this article

HO CHI MINH CITY OPEN UNIVERSITY JOURNAL OF SCIENCE - ENGINEERING AND TECHNOLOGY is currently edited by Nguyen Thuan

More articles in HO CHI MINH CITY OPEN UNIVERSITY JOURNAL OF SCIENCE - ENGINEERING AND TECHNOLOGY from HO CHI MINH CITY OPEN UNIVERSITY JOURNAL OF SCIENCE, HO CHI MINH CITY OPEN UNIVERSITY
Bibliographic data for series maintained by Vu Tuan Truong ().