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

• Published in: Applied microbiology and biotechnology 2019-06, Vol.103 (12), p.4779-4788
• Main Authors: Kim, Young Su, Karisa, Nadia, Jeon, Woo Young, Lee, Hongweon, Kim, Yeu-chun, Ahn, Jungoh
• Format: Article
• Language: English
• Subjects: Affinity; Affinity chromatography; Bacteria; Batch Cell Culture Techniques; Batch culture; Biomarkers; Biomarkers - blood; Biomedical and Life Sciences; Biotechnological Products and Process Engineering; Biotechnology; Brain; Brain natriuretic peptide; Cation exchange; Cation exchanging; Cell culture; Cell density; Chemiluminescence; Chromatography; Chromatography, Affinity; Congestive heart failure; Diagnosis; E coli; Enzyme-Linked Immunosorbent Assay; Enzymes; Epitopes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fed-batch culture; Heart failure; Heart Failure - blood; Heart Failure - diagnosis; Humans; Immunoassay; Levels; Life Sciences; Luminescent Measurements; Microbial Genetics and Genomics; Microbiology; Monoclonal antibodies; Natriuretic Peptide, Brain - biosynthesis; Organic chemistry; Peptide Fragments - biosynthesis; Peptides; Physiological aspects; Protease; Proteinase; Recombinant Proteins - biosynthesis; Tobacco; Viruses; Wet cells
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-019-09826-8

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 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.