当前位置： SCI文献检索 > APPLIED MICROBIOLOGY AND BIOTECHNOLOGY期刊下所有文献 > High-level production of N-terminal pro-brain natriuretic peptide, as a calibrant of heart failure diagnosis, in Escherichia coli.

High-level production of N-terminal pro-brain natriuretic peptide, as a calibrant of heart failure diagnosis, in Escherichia 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 the N terminus 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 commercial monoclonal 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.

journal_name

Appl Microbiol Biotechnol

journal_title

Applied microbiology and biotechnology

authors

Kim YS,Karisa N,Jeon WY,Lee H,Kim YC,Ahn J

doi

10.1007/s00253-019-09826-8

subject

Has Abstract

pub_date

2019-06-01 00:00:00

pages

4779-4788

issue

12

eissn

0175-7598

issn

1432-0614

pii

10.1007/s00253-019-09826-8

journal_volume

103

pub_type

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