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

Fasehee, H. and Rostami, A. and Ramezani, F. and Ahmadian, G. (2018) Engineering E. coli cell surface in order to develop a one-step purification method for recombinant proteins. AMB Express, 8 (1).

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Abstract

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. © 2018, The Author(s).

Item Type: Article
Additional Information: cited By 0
Uncontrolled Keywords: chimeric protein; metallothionein; primer DNA; recombinant protein; sortase, amino acid sequence; Article; bacterial strain; carboxy terminal sequence; cell fractionation; cell surface; Escherichia coli; gene expression; genotype; molecular docking; molecular dynamics; nonhuman; plasmid; polyacrylamide gel electrophoresis; protein cleavage; protein purification; Western blotting
Subjects: QV Pharmacology
Depositing User: eprints admin
Date Deposited: 25 Dec 2018 15:14
Last Modified: 26 Jun 2019 09:37
URI: http://eprints.iums.ac.ir/id/eprint/6152

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