Calibrator for NT-proBNP immunoassay
The invention provides a method to efficiently express high levels of a recombinant untagged NT-proBNP for use as a calibrator in NT-proBNP immunoassays.
1. A stepwise method for expression of NT-BNP (N-terminus brain natriuretic peptide) in Escherichia coli ( E. coli ) without an N-terminal histidine affinity chromatography tag for purification comprising sequentially:
a) introducing SEQ ID NO:1 at the 5′ end of a nucleic acid encoding for NT-BNP;
b) replacing codons 3 through 12 of said nucleic acid encoding for NT-BNP of step a with SEQ ID NO:2;
c) deleting codons 1 and 2 of said nucleic acid encoding for NT-BNP of step b; and
d) transfecting an E. coli culture with the nucleic acid of step c wherein said E. coli culture then expresses NT-BNP without said affinity chromatography based tag for purification.
2. The method of claim 1 further comprising purifying said NT-BNP from said E. coli culture.
3. The method of claim 2 wherein said purifying is carried out by fast/performance liquid chromatography (FPLC).
4. The method of claim 1 wherein the NT-BNP is an N-terminal fused peptide that comprises a mutated phage T7 gene 10-translation initiation region and does not include any other N-terminus affinity chromatography tag for purification.
5. A method for expressing a NT-BNP (N-terminus brain natriuretic peptide) in Escherichia coil ( E. coli ) without an N-terminal histidine affinity chromatography tag for purification, the method comprising:
a) constructing an expression plasmid, the expression plasmid comprising, in contiguously linked sequence, SEQ ID NO:1, SEQ ID NO:2, and the nucleic acid sequence encoding N-terminal brain naturetic peptide from codon 13 to codon 76; and
b) transfecting an E. coli culture with the expression plasmid of step a wherein said E. coli culture then expresses NT-BNP.