Cloning and Expression of Fructosyl-amino Acid Oxidase Gene fromCorynebacteriumsp. 2-4-1 inEscherichia coli

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.

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

Biochemistry (Moscow) ◽

10.1134/s0006297909020035 ◽

2009 ◽

Vol 74 (2) ◽

pp. 137-144 ◽

Cited By ~ 10

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

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New Enzymatic Assay for Glycohemoglobin

Clinical Chemistry ◽

10.1373/49.2.269 ◽

2003 ◽

Vol 49 (2) ◽

pp. 269-274 ◽

Cited By ~ 28

Author(s):

Ikunosuke Sakurabayashi ◽

Tatsurou Watano ◽

Satoshi Yonehara ◽

Kaori Ishimaru ◽

Kaoru Hirai ◽

...

Keyword(s):

Amino Acid ◽

Blood Cells ◽

Hplc Method ◽

Enzymatic Assay ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Blood Samples ◽

Total Hemoglobin ◽

Patients With Diabetes ◽

Fructosyl Amino Acid Oxidase

Abstract Background: Previous methods to measure glycohemoglobin (GHb) have been time-consuming or imprecise; we therefore developed a new enzymatic assay for GHb. Methods: Blood cells were first hemolyzed, and hemoglobin was digested with protease to yield fructosyl amino acid. Fructosyl amino acid oxidase acts on the fructosyl amino acid and generates hydrogen peroxide, which reacts with chromogens in the presence of peroxidase. Total hemoglobin was measured spectrometrically in the same reaction tube. The results were reported as the ratio of the concentrations of GHb and hemoglobin. Results: The measured values were comparable to those determined with a HPLC method and with an immunoassay in blood samples from 2854 patients with diabetes. Regression analysis for the enzymatic assay (y) vs the HPLC method (x) produced the following: r = 0.979; slope, 0.994 [95% confidence interval (CI), 0.986–1.001]; y-intercept, 0.04% (95% CI, −0.09% to 0.01%); n = 2854. For the enzymatic assay (y) vs the immunoassay (x), the regression statistics were as follows: r = 0.982; slope, 1.002 (95% CI, 0.995–1.009); y-intercept, 0% (95% CI, −0.05% to 0.05%); n = 2854. Conclusions: The values measured by the new enzymatic assay are sufficiently correlated with those of the conventional HPLC method and immunoassay, but the proposed assay for GHb is rapid and has high precision.

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Review of Fructosyl Amino Acid Oxidase Engineering Research: A Glimpse into the Future of Hemoglobin A1c Biosensing

Journal of Diabetes Science and Technology ◽

10.1177/193229680900300324 ◽

2009 ◽

Vol 3 (3) ◽

pp. 585-592 ◽

Cited By ~ 36

Author(s):

Stefano Ferri ◽

Seungsu Kim ◽

Wakako Tsugawa ◽

Koji Sode

Keyword(s):

Amino Acid ◽

Hemoglobin A1c ◽

Point Of Care ◽

Detection Methods ◽

Future Research ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Engineering Research ◽

Glycated Proteins ◽

Fructosyl Amino Acid Oxidase

Glycated proteins, particularly glycated hemoglobin A1c, are important markers for assessing the effectiveness of diabetes treatment. Convenient and reproducible assay systems based on the enzyme fructosyl amino acid oxidase (FAOD) have become attractive alternatives to conventional detection methods. We review the available FAOD-based assays for measurement of glycated proteins as well as the recent advances and future direction of FAOD research. Future research is expected to lead to the next generation of convenient, simple, and economical sensors for glycated protein, ideally suited for point-of-care treatment and self-monitoring applications.

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