[Frontiers in Bioscience 16, 2128-2143, June 1, 2011]

Identification of the Lactobacillus SLP domain that binds gastric mucin

Zhihua Liu1, Tongyi Shen2, Mary Pat Moyer3, Huanlong Qin1

1Department of Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, 200233, China, 2Department of Surgery, Shanghai Tenth People's Hospital, Shanghai, 200072, China, 3INCELL Corporation, San Antonio Texas, 78249, United States of America

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Protein expression system and reagents
3.2. Culture of L. plantarum and isolation of SLPs
3.3. Labeling of porcine gastric mucin with HRP
3.4. Identification and verification of proteins and peptides responsible for adhesion to the gastric mucin
3.4.1. Preliminary screening of SLP(s) adhering to gastric mucin by SDS-PAGE and Western blotting
3.4.2. Detection of the protein adhering to the gastric mucin by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)
3.5. Identification of the protein adhering to gastric mucin by cloning and expression of genes encoding the putative target proteins
3.5.1. Cloning of genes encoding the potential target proteins in E coli
3.5.2. Expression of pET32-target genes in E. coli and purification of the expressed proteins
3.5.3. Confirmation of the ability of the target protein to adhere to the gastric mucin
3.5.4. Identification of the smallest domain within IMP-2 responsible for adhesion to the intestinal epithelium
3.5.5. Construction of plasmid vectors
3.5.6. Expression and purification of the mature IMP-2 and its truncated forms encoded by the plasmids
3.5.7. Identification of the ability of recombinant IMP-2 forms to adhere to the gastric mucin by Western blotting with ECL
3.5.8. Expression and purification of MIMP
3.5.9. Preparation of polyclonal anti-MIMP antibodies and identification by Western blotting with ECL and enzyme-linked immunosorbent assay
3.5.10. Detection of IMP isolated and purified from L. plantarum by polyclonal anti-MIMP antibodies
3.6. Confirmation of the ability of the purified MIMP to competitively adhere to intestinal epithelial cells
3.7. Flow cytometry for determining dendritic cell (DC) maturation
3.8. Statistical analysis
4. Results
4.1. Identification of IMP-2
4.1.1. Detection of the SLP of L. plantarum adhering to the gastric mucin
4.1.2. Cloning, expression and purification of the potential target proteins in E. coli
4.2. Identification of MIMP
4.2.1. Primary amino acid sequence analysis of IMP-2
4.2.2. Cloning and expression of gene sequences encoding the mature or truncated forms of IMP-2 and purification of the recombinant proteins
4.2.3. Identification of the ability of recombinant IMP forms to adhere to the gastric mucin by SDS-PAGE and Western blotting with enhanced chemiluminescence (ECL)
4.2.4. Recognition of polyclonal anti-MIMP antibodies with the recombinant MIMP
4.2.5. Detection of IMP isolated and purified from L. plantarum with anti-MIMP polyclonal antibodies
4.3. MIMP competitively adheres with EIEC/EPEC to NCM460 cells
4.4. MIMP adheres to and maturates iDCs as detected by flow cytometry
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

Surface layer proteins (SLPs) of lactobacillus bacteria have some structural regions responsible for adhesion to the intestinal epithelium. To identify the SLP and the smallest domain within the protein that is responsible for the adhesion of the bacterium to the intestinal epithelium, L. plantarum strain CGMCC1258 was investigated in this study. Using bioinformatics and molecular techniques, we first identified and purified a novel protein, integral membrane protein-2 (IMP-2, 33-45 kDa) responsible for adhesion to gastric mucin. Truncated forms of IMP-2 were then constructed and expressed, and the amino acids from 515 to 575 (designated micro IMP, MIMP) was identified as the smallest domain responsible for adhesion to gastric mucin. Competing assay was performed, which further confirmed the ability of MIMP to compete with enteroinvasive E. coli and enteropathogenic E. coli to adhere to cells of a normal colon cell line, NCM460. Furthermore, MIMP could maturate dendritic cells. These findings set a foundation for further investigation on the role of MIMP in the treatment and prevention of inflammation-related diseases of the intestine.