In vivo and in vitro cleavages of inteins through modifications of terminal regions and revisions of environmental factors

Abstract

A controllable cleavage reaction of inteins to release target proteins from affinity tags is an ideal method for the production of recombinant proteins. A precursor protein, consisting of a GyrA intein flanked by a cellulose binding domain fused at its N-terminus and human basic fibroblast growth factor (bFGF) fused at the C-terminus, was expressed initially. Then three amino acid substitutions including Cys1Ala, Ser179Gly and His197Gln were conducted on the Mxe GyrA intein part of this precursor via DNA mutagenesis. It is interesting to see that the precursor protein was rapidly cleaved at the intein's C-terminus in the mutant of Ser179Gly; this precursor protein is shown to be expressed in increased folds of amounts when a double amino acid substitution, Cys1Ala and His197Gln, was performed. The mutated protein was able to bind to Avicel (a microcrystalline cellulose preparation). Thus, bFGF was efficiently cleaved from the precursor protein’s GyrA intein when incubated with 50 mM Tris-HCl at pH 8.5, or 100 mM succinic acid and 10 mM CaCl₂ at pH 6.3. Both in vitro cleaved bFGF proteins proved to be authentic and bio-active through analysis of liquid chromatography tandem mass spectrometry (LC-MS/MS) and MTT assays. A systematic purification protocol for purifying bFGF, with the use of a separate 3-step approach involving cation exchange chromatography, followed by heparin affinity chromatography and gel filtration, is next introduced in this study, and a major portion of abFGF (~85%) present in the cell lysate was successfully purified. Besides, the purification and in vitro induced cleavage methods for harvesting two other recombinant proteins, hEGF and hGH, are herein reported. Key Words: Mxe GyrA intein, Mutagenesis, Cellulose binding domain, bFGF, S179G, H197Q, C1A, Succinic acid, CaCl₂

Date of Award	2019
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology