A practical approach to unveiling auto-catalytic cleavages mediated by Mxe GyrA intein and improving the production of authentic bFGF

Abstract

A controllable cleavage activity of inteins to release target proteins from affinity tags is an attractive approach for the production of heterologous proteins. Expression of CellBD-GyrA-bfgf (CGF), a fusion precursor comprising the cellulose binding domain (CellBD) of an endoglucanase (Eng) encoded by the cenA gene of Cellulomonas fimi at the N-terminus, Mxe GyrA intein (GyrA) in the middle, and human basic fibroblast growth factor (bfgf) at the C-terminus, resulted in incomplete auto-catalytic cleavages and a partial release of free bFGF in the cytosol of Escherichia coli. Amino acid substitutions involving Cys1Ala (C1A) and His197Gln (H197Q) performed simultaneously at both termini of GyrA on CellBD-GyrA-bfgf, thus forming the 49-kDa full-length CGF (H197Q; C1A) mutant precursor (49-kDa MP), were found to exhibit an inhibitory effect on the cleavage detected between CellBD and GyrA, thereby enhancing the reservation of intact 49- kDa MP, which was shown to bind well to Avicel. Sequencing analysis showed that 49-kDa MP underwent complex auto-processing activities involving self-cleavages, proteolysis and fusion of degraded fragments to give rise to a variety of smaller intermediates. One such intermediate, 39-kDa (SI), was also found to be able to bind to Avicel. Free bFGF was effectively cleaved from 49-kDa MP and 39-kDa SI bound on Avicel at pH 8.5 or in the presence of succinic acid. Released bFGF was substantiated to possess the authentic structure and potent bioactivity. The findings may pave way for the development of a facile scalable approach for enhancing production of a heterologous protein. Keywords: Mxe GyrA Intein; Site-Specific Mutagenesis; Cellulose Binding Domain; Avicel; bFGF ; in vitro Cleavages; pH 8.5 ; Succinic Acid