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

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.

Identification of novel immunogenic proteins against Streptococcus parauberis in a zebrafish model by reverse vaccinology.

Streptococcus parauberis is the major infectious agent of streptococcosis in the olive flounder (Paralichthys olivaceus), causing serious economic damage. In this study, we identified potential vaccine candidates against S. parauberis by reverse vaccinology. In total, the 2 out of 21 proteins were identified as vaccine candidates from two available S. parauberis genomes. The membrane-anchored protein SEC10/PgrA and the metal ABC transporter substrate-binding lipoprotein mtsA were potent antigenic proteins based on western blotting with mouse-derived antiserum against whole bacteria of S. parauberis serotypes I and II. In particular, metal ABC transporter substrate-binding lipoprotein (mtsA) showed similar protective immunity to that of whole-cell bacterins against S. parauberis in a zebrafish model. These results suggest that mtsA may be considered as a novel candidate in the development of vaccines against S. parauberis.