Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase

2009 ◽  
Vol 74 (2) ◽  
pp. 137-144 ◽  
Author(s):  
A. Gholizadeh ◽  
B. B. Kohnehrouz
Keyword(s):  
Escherichia Coli ◽  
Zea Mays ◽  
Amino Acid ◽  
Molecular Cloning ◽  
Zea Mays L ◽  
Cloning And Expression ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Expression In Escherichia Coli

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

2021 ◽  
Vol 11 (1) ◽  
pp. 21-29
Author(s):  
Ho Ta Giap ◽  
Phan Ngoc Han ◽  
Tran Le Duy Phuong ◽  
Phung Thi Thu Phung ◽  
Vu Van Van
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Exchange Chromatography ◽  
Cloning And Expression ◽  
Future Application ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Colony Pcr ◽  
E Coli ◽  
Fructosyl Amino Acid Oxidase

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.

Optimization of human d-amino acid oxidase expression in Escherichia coli

2009 ◽  
Vol 68 (1) ◽  
pp. 72-78 ◽  
Author(s):  
Diego Romano ◽  
Gianluca Molla ◽  
Loredano Pollegioni ◽  
Flavia Marinelli
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Expression In Escherichia Coli
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