Screening And Expression Of Antmicrobial Peptide Gene From CDNA Library Of Pteromalus Puparum

More and more research focused on the developing of new classes of antimicrobial peptides (AMPs) with particular biological activity and novel mechanisms of action. We devoted ourselves to find a new approach that would greatly simplify and accelerate the process to isolate novel AMPs. The ability of the new method for library screening makes it a high throughput tool for mining huge numbers of DNA sequences for possible AMP coding sequences, as well as DNA sequences, whose transcriptional, translational, as well as metabolic products, are toxic to bacteria. This capacity is particularly useful in isolating antimicrobial genes whose products occur at low abundance levels, or in organisms that are not easily available, thus difficult to be discovered by traditional protein chemistry methods.1. We constructed a cDNA expression library using mRNA from the Ptermalus puparum, and screened the cDNA library for DNA sequences having antimicrobial activity. After nucleotide sequencing and sequence alignment, we find the sequence search did not generate much homology to its own DNA sequences deposited in the public databases. Nevertheless, sequence of clone PP30 is found to contain a domain that is 50% homologous to antibacterial peptide abaecin from honey bee (Casteels et al.,1990). In addition, three isolated clones contain sequences coding for ribosomal proteins, not yet known to be AMPs.2. Four short peptides were synthesized chemically and tested against both Gram-negative and-positive bacteria. The four peptides exhibited various potencies of antibacterial activities against both Gram-negative and Gram-positive bacteria, with MIC ranging from 8.0μM for PP13 against S. lutea to 166.7μM for PP102 against E. coli. To assess the cytotoxicity of the four peptides against mammalian cells, the percentage of hemolysis was measured against rat red blood cells at various concentrations (0-250μM). After 1 h of incubation, a low level of hemolytic activity was observed at peptide concentrations higher than 125μM. The results indicate that these peptides are relatively safe to mammalian cells. Like many other AMPs, the bactericidal activity of the four peptides against E. coli decreased with increasing concentration of NaCl.3. Transmission electron microphotographs of the PP13-treated E. coli cells indicate that PP13 treatment caused remarkable cell structural changes and extensive damage of cell membranes. Compared to the untreated E. coli cells, PP13-treated cells show abnormal morphologies, such as cell rapture and leakage of cell contents. It indicated that the bacterial cell membrane is a target of PP13.4. CD experiments on the four peptides were performed in 100μM phosphate buffer (pH 7.4), as well as in 50% TFE and 25 mM SDS. All the peptides adopted a random conformation as a major structure in the phosphate buffer. PP13 adoptedα-helical structure in the presence of either TFE or SDS micelles. PP113 adopted a well definedα-helical structure in the presence of 50% TFE. PP102 and PP113 adopted a random conformation as a major structure in the presence of SDS micelles.5. PP30 was successfully sub-cloned into prokaryotic expression vector pTRX, forming the recombinant expression vectors, pTRX-PP30, then introduced into E.coli BL21 (DE3) for expression. In the pTRX/BL21 system, His-PP30 fusion protein was high expressed. The fusion proteins were confirmed by Western blotting analysis with anti-His-antibody.