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

Cited 0 time in webofscience Cited 0 time in scopus
  • Hit : 23
  • Download : 0
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 (97.5mg/l of bacterial culture; 3.25mg/g of wet cell) was 55-fold higher than that reported in previous studies (0.5-1.75mg/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 r(2)=0.995). These results demonstrate the efficiency of our process for mass producing (gram-to-liter level) rNT-proBNP with acceptable analytical performance.
Publisher
SPRINGER
Issue Date
2019-06
Language
English
Article Type
Article
Citation

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, v.103, no.12, pp.4779 - 4788

ISSN
0175-7598
DOI
10.1007/s00253-019-09826-8
URI
http://hdl.handle.net/10203/262736
Appears in Collection
CBE-Journal Papers(저널논문)
Files in This Item
There are no files associated with this item.

qr_code

  • mendeley

    citeulike


rss_1.0 rss_2.0 atom_1.0