Molecular cloning, expression and adhesion analysis of silent slpB of Lactobacillus acidophilus NCFM

Daodong Pan,Yao Yang,Zhen Wu,Fawze Nadari,Yuxing Guo,Xiaoqun Zeng,Xiangyue Li

doi:10.1186/s13568-018-0631-2

Abstract

The slpB gene of Lactobacillus acidophilus NCFM, which differs from the slpA gene and is silent under normal conditions, was successfully amplified and ligated to the corresponding available sites on a recombinant pET-28a vector. Then the pET-28a-slpB vector was transformed into Escherichia coli DH (DE3) and the fusion His-slpB protein was expressed by induction with 1 mM IPTG for 14 h at 37 °C. The resulting His-slpB protein (SB) had a relative molecular weight of 48 kDa. It was purified using a Ni-NTA column and was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot contrastive analysis. The slpA protein (SA) from L. acidophilus NCFM was extracted and purified. It had a relative molecular weight of 46 kDa. Circular dichroism measurements suggested that the two S-layer proteins had a high β-sheet content and a low α-helix structure content. In an adhesion experiment, SA displayed higher adhesive capability towards Caco-2 cells than did SB. The results suggest that these two S-layer proteins could have biotechnological applications.

Highlights

Probiotic microorganisms, such as Lactobacillus, which is a normal flora found in the intestines of humans and animals (Walter et al 2003), balance endogenous bacteria, protect the intestinal tract from competition, manufacture antimicrobial molecules, and stimulate mucosal immunity (Singh et al 2016)
NCFM showed that the gene was pure, complete and suitable to use to clone the slpB gene (Fig. 1a)
The pET-28a vector was digested with NdeI/XhoI restriction enzymes and the slpB gene was ligated to the corresponding sites on the pET-28a vector to form a recombinant pET-28a-slpB expression vector (Fig. 1c)

Summary

Introduction

Probiotic microorganisms, such as Lactobacillus, which is a normal flora found in the intestines of humans and animals (Walter et al 2003), balance endogenous bacteria, protect the intestinal tract from competition, manufacture antimicrobial molecules, and stimulate mucosal immunity (Singh et al 2016). S-layer proteins are used in the efficient expression and localization of heterologous proteins or polypeptides because of the high transcriptional efficiency of gene promoters (Lindholm et al 2004). L. acidophilus has two S-layer protein genes (Boot et al 1993): slpA is expressive under normal conditions, while slpB is silent. These two S-layer proteins encoding genes are located on a chromosomal fragment of 6 kb (the slp segment) (Boot et al 1996). It should be possible to create an expression system to express the slpB gene This endeavor is important in order to analyze the biological function of the silent protein

Objectives

Methods

Results

Conclusion