Engineering E. coli cell surface in order to develop a one-step purification method for recombinant proteins

被引:0
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作者
Hamidreza Fasehee
Amin Rostami
Fatemeh Ramezani
Gholamreza Ahmadian
机构
[1] National Institute of Genetic,Department of Industrial and Environmental Biotechnology
[2] Engineering and Biotechnology,Protein Science Group, Gastroenterology and Liver Disease Research Center, Research Institute for Gastroenterology and Liver Diseases
[3] Shahid Beheshti University of Medical Sciences,Physiology Research Center, Faculty of Medicine
[4] Iran University of Medical Sciences,undefined
来源
AMB Express | / 8卷
关键词
Surface display; Sortase; Molecular dynamic; Protein purification;
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摘要
Sortases are enzymes mostly found in Gram-positive bacteria which cleave proteins site-specifically. This feature makes them a promising tool in molecular biology and biotechnology. In this study, using bacterial surface display of recombinant proteins and ability of sortase A in site-specifically cleavage of the amino acid sequences, a novel method for one-step purification of recombinant proteins was developed. Using computational program tools, a chimeric protein containing a metallothionein (mt) and chitin binding domain (ChBD) was attached to the C-terminal domain of the truncated outer membrane protein A (Lpp′-ompA) using sortase recognition site (amino acid residues: LPQTG) as a separator. The structure of the chimeric protein was simulated using molecular dynamics to determine if the LPQTG motif is accessible to the sortase active site. The designed chimeric protein was expressed and purified. The purified chimeric protein was also displayed on the surface of E. coli cells. Both purified chimeric protein and the E. coli cells displaying Lpp′-ompA-mt-ChBD carrier protein were then treated with sortase to evaluate the efficiency of sortase-mediated cleavage of purified chimeric protein as well as surface displayed-chimeric protein. It is shown that mt-ChBD protein was successfully cleaved and dissociated from Lpp′-ompA carrier and released into the medium after treatment with sortase in both recombinant protein and surface displayed-chimeric protein. The experimental results confirmed the molecular dynamics analysis results. The presented method could be regarded as a novel strategy for one step expression and purification of recombinant proteins.
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