Designing And Construction Of The Recombinant Mutant Of Human Endotoxin Binding Peptide

Background and objectiveLipopolysaccharide binding protein (LBP) is a vital carrier protein molecular which can bind with Lipopolysaccharide (LPS) and its C-terminal binds the CD14, then forms a triad-complex of LPS-LBP-CD14 and triggers TLR4-MD2-MyD88 complex to initiate intracellular signal transduction, so as to lead the activation of nuclear factor-kB (NF-kB) and cytokine release. Many studies have shown that the derived peptides of natural proteins, such as LBP, BPI and LALF, have the ability of binding and neutralizing entodoxin, then inhibit LPS-induced inflammatory response in vitro and vivo. At the same time, they have a common feature that the domain of those natural proteins binding with LPS are rich in positive charge and hydrophobic amino acids. The molecular structure of LPS contains negatively charged phosphate groups and hydrophobic long-chain fatty acids, which are easily combined with a positively charged and hydrophobic molecular. Therefore, by replace of the amino acids, Increasing the number of positive charge and hydrophobicity may lead to increase the activity of peptides to resist endotoxin. However, different amino acids have different electric charge and hydrophobicity, any replacement of a residue of amino acid can change the charge and hydrophobicity of the peptide, even its spatial structure. So we need to understand which residues of amino acids in these peptides are the critical sites to combine LPS, and select other sites except the critical one to mutate. That is to say, we replace the non-critical amino acids with the one of hydrophobicity, basicity and importance. As we know, the NH2-terminal part of LBP (NH2-LBP) is the binding site of LPS, especially the region of 108-133 amino acides which is rich in hydrophobic and basic amino acids, has a high affinity with LPS containing the negatively charged phosphate groups and long hydrophobic chain fatty acid. In previous study, our team has confirmed the ability of LBP108-133 named endotoxin binding peptide (EBP) to bind and neutralize LPS. Therefore, based on the structural characteristics of LPS and our previous study, we reconstruct the EBP genes and mutate its bases which might influent its biological activity. EBP25 and its mutants were cloned, expressed and purified to study their function for anti-endotoxin activity detection.MethodsUsing the pET-30-LBP recombinant plasmid as a template, EBP25 genes were amplified by PCR and inserted into the prokaryotic expression vector pET-30. The recombinant plasmid pET-30-EBP25 was transfected into E.coli. DH5α, and identified by DNA sequencing.Then the corresponding bases of the 2th valine, the 5th glutamine, the 15th phenylalanine, the 18th asparagine and the 22th aspartic acid were replaced by the corresponding bases of lysine, lysine, tryptophan, lysine, alanine with Quikchange site-directed mutation. The mutant of recombinant plasmid pET-30-mEBP25 was identified by DNA sequencing too .Then the both EBP25 and mEBP25 were transfected into E.coli. BL21(DE3) PlysS and expressed under IPTG induction. The expression was analyzed by western blotting and purified with His-Tag affinity chromatography.Results1. Construction of the recombinant plasmid pET-30-EBP25: The 96 bp EBP25 genes were amplified by PCR with template of pET-30-NH-LBP, and inserted into the prokaryotic expression vector pET-30. The recombinant plasmid pET-30-EBP25 was constructed and identified by endonuclease and DNA sequencing.2. Acquirement of mutants of pET-30-mEBP25: Using pET-30-EBP25 as template, the corresponding bases of the 2th valine, the 5th glutamine, the 15th phenylalanine, the 18th asparagine and the 22th aspartic acid were replaced by the corresponding bases of lysine, lysine, tryptophan, lysine, alanine with Quikchange site-directed mutation.3. Expression and identification of these fusion proteins: The five recombinant plasmids were transformed into E.coli BL21(DE3) plysS, in which EBP25 and m1EBP25 were highly expressed as form of inclusion bodies, while the other three mutants were low or not detectable. The result of SDS-PAGE electrophoresis showed that the newborn protein of EBP25 and m1EBP25 were detected at about 8 KD, identified with the expected fusion protein molecular weight. The specificity of binding with mouse anti-His monoclonal antibody was verified by western blot.4. Purification and detection of these fusion proteins: After separating the dissolved inclusion bodies, these fusion proteins were purified by Invitrogen's Ni-NTA agarose for 6xHis Tag affinity chromatography. EBP25 and m1EBP25 were obtained after dialysis.Then the rate of purity was 96.8% tested by mass spectrum.ConclusionsWe gained the fusion expression vetors of pET-30-EBP25 and its mutant of pET-30-m1-5EBP25, selected and purified the highly expression of recombinant proteins EBP25 and m1EBP25, which can be used to study the biological function of neutralizing endotoxin/lipopolysaccaride.