title: Direct site-specific immobilization of protein A via aldehyde-hydrazide conjugation.
abstract: Immobilization of affinity ligands on supporting matrices is a key step for the preparation of affinity chromatography resins, and an efficient coupling strategy can significantly improve the validity and cost of the affinity system, especially for systems that employ expensive recombinant proteins or antibodies as affinity ligands. This study described a simple method for obtaining site-specific immobilization of protein A (the ligand) via aldehyde-hydrazide conjugation and its application in antibody purification via protein A chromatography. An aldehyde group was generated at the N-terminus of protein A in vivo by co-expressing a formylglycine-generating enzyme (FGE) and recombinant protein A containing a FGE recognizing sequence (aldehyde tag) in Escherichia coli. The resulting aldehyde allowed direct immobilization of protein A onto the hydrazide-modified agarose matrices under mild condition. We found that 100mM aniline was most effective for catalyzing the coupling reaction, and the recombinant protein A could be coupled with high selectivity, directly from a crude cell extract. The site-specific immobilized protein A showed good capacity for antibody purification. The specificity of the aldehyde-hydrazide reaction not only allowed site-specific immobilization of affinity ligands, but also improved the cost of the process by employing unpurified ligands, a method that might be of great use to industrial applications.
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116023, PR China.
authors.forename: Berlin
authors.initials: B
authors.lastname: Zang
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116023, PR China. Electronic address: renjun@dlut.edu.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Ren
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116023, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116023, PR China. Electronic address: lyj81@dlut.edu.cn.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Jia
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D013205:Staphylococcal Protein A / Q000737:chemistry*
chemical_list: D000447:Aldehydes; D001386:Azides; D013205:Staphylococcal Protein A
keywords: Aldehyde-tag; Formylglycine-generating enzyme; Ligand immobilization; Protein A chromatography; Site-specific immobilization