Identification And Characterisation Of Domains Responsible For S-protein Cell Wall Binding Of Lactobacillus Acidophilus CICC 6074

Lactic Acid Bacteria,LAB is an important probiotic in the human intestine and can regulate the body’s immune function.S-layers protein(SLP)is a superficial protein that exists outside the cell membrane or cell wall and is an important adhesion factor for lactic acid bacteria colonizing the intestine.At present,the functional domains of S-layer protein anchors of different strains are not perfect and clear,so the study of their domain is of great significance.The S-layer protein gene of Lactobacillus acidophilus CICC 6074 was cloned and the amino acid sequence homology and physicochemical properties of S-layer protein were analyzed to predict its domain.Cloned and expressed of L.acidophilus CICC 6074 C-terminal truncation sequence of the S-layer protein and synthesied the N-terminal sequence of the S-layer protein were incubated with the L.acidophilus CICC 6074 cell wall to study the cell wall anchoring domain of the S-layer protein.The specific research content and results are as follows:Firstly,the L.acidophilus CICC 6074 and NCFM S-layer proteins were treated with acidic LiCl and purified by Sephadex G-75 gel chromatography.The molecular weights of both proteins were 46 kDa as determined by SDS-PAGE,which was in accordance with the size range of the slpA gene expression products.A 1300 bp SlpA gene band was amplified from L.acidophilus CICC 6074 by PCR and had 99%sequence similarity to the L.acidophilus NCFM SlpA gene.L.acidophilus CICC 6074 S-layer proteins amino acids was compared with L.acidophilus NCFM,L.crispatus JCM 5810 and L.helveticus JCM 1003 in the database using Clustal W,via Genetyx,ProtParam,TMHMM and PSIPRED biological software revealed that the C-terminus of this S-layer protein is extremely conserved and its similarity is as high as 77%or more.It has a large number of highly hydrophobic amino acids.Hydrophobic and hydrophobic amino acids are alternate distributed SLP has a transmembrane structure,the isoelectric point is 9.59.The S-layer protein is predicted to have two functional domains,namely cell wall anchoring domains and self-assembling domains.Secondly,through the genetic engineering technology,the gene SlpA encoding the L.acidophilus CICC 6074 S-layer protein and its C-terminal truncated fragment were respectively fused with green fluorescent protein(GFP)to construct a recombinant vector and induced in E.coli,expressed and purified by His-Tag protein.The results of SDS-PAGE showed that four fusion proteins(S-layer full-length GFP-SA and C-terminally truncated GFP-SAC,GFP-SAC1,and GFP-SAC2)were successfully purified,and the molecular weights of the four fusion proteins were respectively GFP-SA(74 kDa)and GFP-SAC(41 kDa)and GFP-SAC1(33 kDa)and GFP-SAC2(34 kDa).Finally,the cell wall CWF of L.acidophilu CICC 6074 was extracted and combined with GFP-SA,GFP-SAC,GFP-SAC 1,GFP-SAC2 and the synthetic N-terminal truncation protein GFP-SNn(n=1-5),respectively.The fluorescence results showed that the L.acidophilu CICC 6074 S-layer protein cell wall anchoring domain is located at its C-terminus.GFP-SAC was incubated with LiCl-treated and untreated L.acidophilu CICC 6074 cells,the fluorescence value of LiCl-treated cells was 1.5-fold higher than that of untreated cells.The fluorescence intensity of the LiCl-treated was significantly lower than that of untreated LiCl-treated cells observed by fluorescence microscope.The results strongly suggested that the L.acidophilu CICC 6074 S-layer protein cell wall anchoring domain is located at its C-terminus.After the removal of the teichoic acid component in CWF with 5%trichloroacetic acid(TCA),the fluorescence response was decreased from 39,424 RFU to 9,261 RFU,and the organic phosphorus content in CWF also decreased by 72%.The results showed that cell wall anchored receptor for S-layer protein of L.acidophilu CICC 6074 is teichoic acid.In summary,this subject cloned and expressed the C-terminal fusion protein of L.acidophilu CICC 6074 S-layer protein.N-terminal truncation sequence of S-layer protein was synthesized.C-terminal fusion protein and N-terminal protein were incubated with the bacterial cell wall,respectively.The cell wall anchoring domain of the S-layer protein was ascertained.The research provides a theoretical basis for the further development and application of L.acidophilus and its S-layer proteins.