Soluble expression of recombinant coagulation factor IX protein using Escherichia coli

Abstract

Hemophilia B is a congenital bleeding disorder caused by factor IX (FIX) deficiency. Generation of recombinant FIX (rFIX) is required for detecting a Hemophilia B indicator, anti-FIX antibody. In this study, we described a method for producing recombinant FIX (rFIX) using Escherichia coli. We constructed a FIX-expressing plasmid without a fusion tag protein-encoding gene and produced rFIX as a soluble form within five days. Dose-dependent curve was obtained from ELISA using anti-FIX antibody, indicating that the rFIX can be used as an antigen to detect anti-FIX antibody with high affinity and sensitivity.

Keywords: Escherichia coli; Factor IX; Hemophilia; Protein expression; Recombinant protein.

Fig. 1

(A) Plasmid DNA maps for the expression of C-terminal His-tag-conjugated FIX (FIX-H) and N-terminal His-tag-conjugated FIX-coding gene (H-FIX). P, TEE, His (H), FXa, MCS, dot, and AmpR indicates primer, translation enhancing element sequence, His-tag, Factor Xa sequence, multiple cloning site, stop codon, and ampicillin resistance gene, respectively; (B) Schematic representation of the entire protein production system; T.F. indicates transformation; (C) SDS-PAGE analysis of FIX-H or H-FIX. An arrow indicates the location of target protein.

Fig. 2

(A) SDS-PAGE analysis of the pET28a-, PGK-conjugated pET28a-, or GST-conjugated pGEX4T1-coded FIX proteins; (B) SDS-PAGE analysis of rFIX proteins and empty pCold-I-coded protein (a pCold-I vector without inserting an FIX-expressing gene) before or after induction; (C) SDS-PAGE analysis of total (T) or soluble (S) fraction of rFIX proteins and empty pCold-I-coded protein after induction. Arrows indicate the location of target protein.

Fig. 3

(A) Western blotting analysis of rFIX proteins and empty pCold-I-coded protein using anti-His-tag antibody; (B) western blotting analysis rFIX proteins and empty pCold-I-coded protein using anti-FIX antibody; (C) SDS-PAGE analysis and western blotting of H-FIX and four laboratory available recombinant proteins. Arrows indicate the location of target protein.

Fig. 4

(A) Schematic representation of the ELISA; (B) ELISA signal obtained from commercial FIX; (C) ELISA signal obtained from H-FIX; (D) ELISA signal obtained from FIX-H. Error bars represent ±1 SD (n = 3). SD = standard deviation.