Screening Of Novel Antimicrobial Peptides Using Directed Evolutionary Techniques And Expression And Purification Of Amps

Antimicrobial peptide generally refers to small molecule peptide with antibacterial activity,which is a promising alternative to antibiotics because of its wide source,diverse sequences and its ability to kill bacteria,fungi,viruses and other pathogens as well as immunomodulatory effects.Novel antimicrobial peptides are usually obtained by direct isolation and purification from organisms,which is a single source and complex to manipulate.Synthetic biology provides the technical basis for the large number of novel antimicrobial peptides to be mined,and high-throughput screening using protein directed evolution techniques can provide a large number of lead antimicrobial peptides for antimicrobial peptide development.The high synthetic cost of antimicrobial peptides is another major challenge limiting their development into clinical drugs.Heterologous expression of antimicrobial peptides by fusion with carrier proteins is a common method in the industrial production of antimicrobial peptides,and fusion expression based on intrinsic peptides has been shown to yield biologically active antimicrobial peptides.In this study,we constructed a random peptide library using protein directed evolution technique and screened the random peptide library under physiologically relevant conditions by bacterial surface display technique;meanwhile,we achieved high expression of antimicrobial peptide fusion protein by commercial E.coli expression strain as vector using intrinsic peptide expression system,and the antimicrobial peptide released after cleavage of fusion protein has certain antimicrobial activity.The main results are as follows:1.Construction of an AMPs activity detection system based on bacterial surface display technologyUsing E.coli DH5α as the host bacterium and p BAD33 containing an arabinose promoter as the vector,a bacterial surface display system containing four components: Lpp anchoring protein,Omp A transmembrane protein,flexible tether chain and C-terminal antimicrobial peptide was constructed.The system was validated using cecropin P1,which is known to have Gram-negative bacterial killing activity,and the results showed that the AMPs activity detection system based on bacterial surface display technology was successfully constructed.2.A random mutation library of short peptides was constructed and a series of novel AMPs were obtained by screeningA random library was generated by PCR amplification of the single-stranded DNA fragment into a double-stranded fragment using a 75 bp high-throughput mutagenesis primer with a simple codon and ligated to the above antimicrobial peptide activity detection system,which showed that the library included random mutations and that the library members were not limited to a set 25 amino acid residues and had a high diversity of sequence lengths.Under arabinose induction conditions,the OD of each transformant in the library was measured at 600 nm,and those transformants with lower OD than the no-load control were selected and sequenced.The DNA sequences of the top 15% of positive transformants were selected and a total of 26 peptide sequences with bacteriostatic activity were identified.When compared with the database of AMPs,all of the screened peptides were found to be novel AMPs.Moreover,the amino acid composition and hydrophobicity distribution of the novel AMPs were different from those of natural AMPs.3.Soluble expression of AMP fusion proteins using an intein fusion expression systemIn this study,six AMPs with different sources and structures were selected,and the N-terminal fusion protein his-SUMO-Intein-AMP and the C-terminal fusion protein AMP-Intein-SUMO-his were constructed,and the plasmid p ET28 a was used as the expression vector and transferred into E.coli BL21 strain for expression.SDS-PAGE results revealed that all six fusion proteins could be solubilized for expression.4.Active AMPs were obtained by cleavage of inteinThe four fusion proteins,his-SUMO-Intein-Lfcin B,his-SUMO-Intein-PG 1, CAD-Intein-SUMO-his,and Lfcin B-Intein-SUMO-his,were obtained by bulk expression and purification of the above AMP fusion proteins.The purified N-terminal fusion proteins were induced to self-cleave by changing the p H value of their buffers,while the C-terminal fusion proteins were induced to self-cleave by adding DTT.The Oxford Cup inhibition circle test of the lysis products showed that CAD and Lfcin B with C-terminal fusion were able to inhibit the growth of Escherichia coli,Salmonella and Staphylococcus aureus effectively.The results of the growth curve assay were consistent with the results of the inhibition circle assay,indicating that the active antimicrobial peptide could be obtained by the cleavage of the intrinsic peptide.