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Title 

High-level production of N-terminal pro-brain natriuretic peptide, as a calibrant of heart failure diagnosis, in Escherichia coli

Authors 

Young Su KimN KarisaWoo Young JeonHong-Weon LeeY C KimJungoh Ahn

Publisher 

Springer Verlag (Germany)

Issue Date 

2019

Citation 

Applied Microbiology and Biotechnology

Keywords 

NT-proBNPBiomarkerDiagnosisHeart failureEscherichia coli

Abstract 

Heart failure (HF) is a coronary disease that affects people worldwide and has a high mortality rate. N-terminal pro-brain natriuretic peptide (NT-proBNP) has been proven to be a useful and accurate biomarker for diagnosing systolic HF. Here, we report a strategy for the high-level production of recombinant (r)NT-proBNP in Escherichia coli. An Fh8 tag with six histidines was fused to theNterminus of NT-proBNP along with the recognition site of tobacco etch virus (TEV) protease; the 6HFh8-NT-proBNP fusion peptide was expressed in flask cultures of E. coli in almost completely soluble form. The peptide was purified by HisTrap affinity chromatography, and the N-terminal tag was cleaved by TEV protease. After a second round of HisTrap affinity chromatography to remove the TEV protease and N-terminal tag, rNT-proBNP was isolated with high purity (≥ 98%) by carboxymethyl cation exchange chromatography. The final yield of purified rNT-proBNP (97.5 mg/l of bacterial culture; 3.25 mg/g of wet cell) was 55-fold higher than that reported in previous studies (0.5?1.75 mg/l of bacterial culture). Furthermore, the high cell density E. coli fed-batch culture enabled high-level production of rNT-proBNP in the order of grams per liter. The purified rNT-proBNP was detected by enzyme-linked immunosorbent assay and chemiluminescence enzyme immunoassay using commercialmonoclonal antibodies recognizing different epitopes, showing a linear dose-response relationship in the range of tested concentrations (slope = 3.58 and r2 = 0.995). These results demonstrate the efficiency of our process for mass producing (gram-to-liter level) rNT-proBNP with acceptable analytical performance.

URI 

https://doi.org/10.1007/s00253-019-09826-8

ISSN 

0175-7598

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-07-10


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