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

Hamidreza Fasehee,Amin Rostami,Gholamreza Ahmadian,Fatemeh Ramezani

doi:10.1186/s13568-018-0638-8

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.

Highlights

Bacterial surface display of heterologous proteins has become an attractive strategy for a wide variety of applications like screening of antibody libraries (Cavallari 2017), as a vaccine delivery vehicle (Kalyanasundram et al 2015), production of cellular adsorbents (Tafakori et al 2012), and development of cellular biosensors (Liang et al 2015)
Results showed that the amount of Mt-Ch in the outer membrane of the cells treated with sortase A (srtA) were decreased comparing to the negative control (Fig. 5)
Analysis of surface expression and cleavage The presence of full-length construct in the supernatant of the surface displaying recombinant bacteria treated with sortase as well as the outer membrane was investigated using western blotting

Summary

Introduction

Bacterial surface display of heterologous proteins has become an attractive strategy for a wide variety of applications like screening of antibody libraries (Cavallari 2017), as a vaccine delivery vehicle (Kalyanasundram et al 2015), production of cellular adsorbents (Tafakori et al 2012), and development of cellular biosensors (Liang et al 2015). In this process, a coding sequence for the passenger protein is fused to the gene encoding a bacterial surface protein. It plays a structural role in the integrity of the bacterial cell surface (Koebnik et al 2000)

Methods

Results

Conclusion