Reconstruction And Fusion Expression And Biological Activity Of Human Cathelicidin LL-37

Bactericidal peptides(anti-bacterial peptide or peptide antibiotics) are small peptides encoded by organism genomic DNA.They have been recognized by their antimicrobial activities in the innate host defense of most living organisms.Most of these peptides consist of 12-60 residues and are less than 10kDa.Antimicrobial peptides play an important role in innate host defense.About 30 cathelicidin members from various mammalian species have been identified.However,only one cathelicidin,hCAP18(human cationic antibacterial protein of 18 kDa) has been found in humans,and its carboxyl-terminal antibacterial peptide,called LL-37,comprises 37 amino acid residues.LL-37 is a a-helical cathelicidin cation peptide owning molecular wieght 4.5kDa.It is showed that LL-37's broad antimicrobial spectrums and not easy to result in drug-resistance.So it is considered to has important applicative value.According to the research,LL-37 contents is lower in vivo of humanbody,and its bactericidal activity is inferior than monkey homologization bactericidal peptide RL-37. Studies on the basis of structure-activity relationship(SAR) manifest that the bactericidal activity and antibacterial spectrum of all a-helical bactericidal peptides correlate with their own positive charge and the number of hydrophobic amino acid residues.Reconstructing the proteinie sequence to increase positive charge and the number of hydrophobic amino acid residues of LL-37 peptide chain is a better pathway to enhance LL-37 antibacterial activity and antiendotoxic activity.In this study,we reconstruct the proteinic sequence of human cathelicidin LL-37 in order to increase its positive charge.The rare codons in the LL-37 gene sequence were substituded by the preferred codons of procaryotic cell,and a fragment of carrier protein molecule(CPM) gene order was added to the N termination of it.The reconstructed LL-37(rLL-37) was inserted into vector pET-30a(+),then the rLL-37 was expressed in E.coli.BL21 Star(DE3) by fusion,in order that it was expressed with high efficiency in procaryotic cell.The rLL-37 was expressed in E.coli.by fusion,which was included specital affinity purification labeling sequence of(FXa) and 6 histidine.We obtained purified rLL-37 after the inclusion bodies of expressed rLL-37 were isolated and purified, desalted,Fxa cutting and so on.At last,the biol-activity of rLL-37 in vivo and vitro was studied.The main results are as following:1.The two-dimensional structure,three-dimensional structure and chemical characters of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL.We obtained reconstructed LL-37 after some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased(Glu₁₆, Asp₂₆,Glu₃₆ of LL-37 were replaced by Gln₁₆,Asn₂₆,Gin₃₆).The static charge of rLL-37 was increased from +5.8 of LL-37 to 9.0 at pH 7.4.Without changing the N termination hydrophobic characterization,the C termination hydrophibic characterization,the two-dimensional and the three-dimensional of LL-37.At the same time,the rare codons in the LL-37 gene sequence were substituded by the preferred codons of procaryotic cell(GGA was replaced by GGT,AGA was replaced by CGT).2.To design a fragment of DNA sequence contained 84 bp,a fragment of carrier protein molecule(CPM) gene order was composed,encoded 28 amino-acid residues and its pHi was 2.7 and net change -6.0 at pH 7.4,then adding the CPM gene order own negetive charge to the N termination of the rLL-37.3.The DNA sequence(210bp) of encoded the CPM and the rLL-37 was obtained successtively by Touch-Down PCR.After the DNA sequence was inserted PMD19-T Simple Vector,and recombined with vector pET-30a(+) again,the expression plasmid pET-30a(+)-CPM-rLL-37 was constructed successtively,which was confirmed by sequence characterization.4.The expression plasmid pET-30a(+)-CPM-rLL-37 was expressed in E.coli BL21 Star(DE3).The expressed fusion protein accounted for 35%of total bacterio-protein,then the expressed product was purified by using affinity binding chromatography with TALON resins successfully.After being cleaved by thrombin fector Xa,the rLL-37 was purified by using high positive ion exchange column Macro-Prep High S successfully.The objective protein of rLL-37 was obtained totally 3.5 mg by Folin-phenol method determination.5.The rLL-37 bactericidal peptide was proved byofinhibitory zone to be able to kill both Gram-negative bacteria and Gram-positive bacteria.By the means of MIC and MBC,it was proved that the rLL-37 had the better bactericidal activity than tradition antibiotic AMP killing Staphylococcus aureus and Bacillus coli XL₁-Blue.The rLL-37 had the same bactericidal activity as tradition antibiotic AMP killing Enterococcus faecalis.6.Through the experimentation of ELISA,the rLL-37 was confirmed that it owned the stronger antiendotoxic activity in vitro.7.Biological activity of the rLL-37 was confirmed without adverse reaction,and it could protect infected mouse to sterilize and relieve the inflammation in vivo.In conclusion,human cathelicidin LL-37 was recreconstruct,and the static charge of rLL-37 was increased from +5.8 of LL-37 to 9.0 at pH 7.4,and the rare codons in the LL-37 gone sequence were substituded by the preferred codons of procaryotic cell.To add the CPM gene order own negetive charge to the N termination of the rLL-37,the static charge of fusion protein was decreased,in order that it was purified by using affinity binding chromatography with TALON resins successfully,and the damage of rLL-37 to expressed host bacterium was decreased as well.At the same time,the rLL-37 polypeptide molecular weight was increased from 8 kDa to 15 kDa.Thereby,its expressive stability and expressive production were enhanced.It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion.Biological activity of the rLL-37 was proved that it is able to kill both Gram-negative bacteria and Gram-positive bacteria,and it owned the stronger antiendotoxic activity in vitro.The rLL-37 was confirmed without adverse reaction, and it could protect infected mouse to sterilize and relieve the inflammation in vivo.It is feasible to produce reconstructed human cathelicidin LL-37 by genetic engineering,which makes it possible to study LL-37 deeply.