High-Level Expression And Purification Of Antimicrobial Peptide Cecropin A-Derivatives In Escherichia Coli And The Preparation Of Antibodies

BACKGROAUNDNowadays,more and more antibiotics have been developed and used to combat with various infectious diseases that threaten the health of humans,but it has been found that many strains of microorganisms are resistant to the commonly used antibiotics. Therefore, it is a hot focus to find novel kinds of antibacterial drug with great developing potential, such as antimicrobial peptide.Most of antimicrobial peptides are small cationic peptides with characteristics of molecular weight<5kDa, heat-resistant, water-soluble, low immunogenicity and rapid action. Both cationic and anionic peptide antibiotics contribute to the innate host defense against a number of microbial pathogens, such as bacterial, fungal pathogens, virus and parasite, which are resistant to conventional antibiotics. Some of them have anticarcinogenesis without affecting human normal cells. Cecropins were the first peptide antibiotics found in 1972 in the pupae of Cecropia (a giant silk moth). By now, antibacterial peptides have been known to exist broadly in insects, plants, animals and human body in vivo. The expression systems of protocaryon, eukaryon and insects have been constructed. Because the antibacterial peptides have antibacterial activity to the procaryotic cell, they are hard to be expressed in engineering bacteria of protocaryon. The low–level expression makes the use of eukaryon expression systems impossible. Compare with the conventional antibiotics, some of their antibacterial activity were poor. The stability in vivo remains to be studied. So we have many things to do, but it is not far away with the further research of antibacterial peptides and transgenic technology.OBJECTIVESTo decrease the antibacterial toxicity to engineering bacteria and to raise the expression production, the tandem repeat of the target peptide fused of cecropin A (CA) to the C-terminus of the 125 codons for the bacterial ketosteroid isomerase (KSI) gene were established. To give more theory evidence for modifying the natural antibacterial peptides and designing the new antibacterial peptides, we design and compare the activity of two a-helicitys of Cecropin A.METHODS1. Analyzed the secondary structure of cecropin A by PredictProtein and the functional sites by PROSITE SCAN, the amino acids sequence of 1 to 19 of CA named as CA19, 18 to 36 as CA36, 2 to 10 and 25 to 32 as CA210 were sclected.2. According to the preference to codon, 3 pairs of reverse complementary nucleotide single strand with a 3-base ATG extension were designed. The phosphorylated oligos were combined and annealinged into 5′-end of double strands. The nucleotide double strands were ligated into tandem repeats by T4 DNA ligase and cloned into pET-31b (+) vector. The pET-31b (+)-CA19 (36, 210)1-5 vectors were transformed into DH5α, the genetic engineered bacteria containing pET-31b (+)-CA19 (36, 210)1-5 were selected by PCR and checked by gel analysis.3. The positive transformed vectors into the E. coli strain BL21 (DE3) were expressed. The optimal inducing conditions to express were deterimined and the expression products were concentrated and identified with Western-blot.4. The expression products were purified by His?Bind chromatography and cleaved into monomer by CNBr, cleaved peptide mixture were purified and vacuum freeze-dried. The purified proteins were identified with Tricine SDS-PAGE.5. The antibacterial activities were investigated by inhibition experiment with Escherichia coli, Enterobacter cloacae, Klebsiella pneumonia, Pseudominas aeruginosa and Staphylococcus aureus.The minimal inhibitory concentrations (MICs) were detected with microamount dilution method. 6.Using the recombinant proteins of CA-derivatives to immune BALB/c mice, blood from the mous tails was collected after four times of immune. Then the antiserums were detected with ELISA assay and Western-blot.RESULTS1. CA19 (36, 210)1-5 and their expression vectors pET-31b(+)-CA19 (36, 210)1-5, were successfully constructed which were confirmed by gel analysis and sequencing.2. The engineered bacteria harboring pET-31b (+)-CA19 (36, 210)1-5 could express the expected tandem fusion proteins when induced by 0.2 mM IPTG, 37℃for 3 h. The CA19 (36, 210)-3 which were the highest level expression in the all five positive recombinants were selected.3. The recombinant proteins were purified by Ni2+-NTA affinity column. SDS-PAGE and Bandscan analysis showed that the target proteins were highly purified and their purity was more than 90%. Monomers were gained by CNBr cleavage and identified with Tricine SDS-PAGE.4. The inhibition experiments showed that the antimicrobial peptide CA19 (36, 210) had the better antibacterial activity to Gram′s negative Escherichia coli, Enterobacter cloacae, Klebsiella pneumonia and Pseudominas aeruginosa that to Gram′s positive Staphylococcus aureus. The activities of the peptides were in the order of CA210>CA19>CA36. The minimum inhibitory concentrations(MICs) of the recombinant proteins against 5 strains were about 16～128μg/ml.5. The titers of CA19, CA36, CA210 after four times of immunes were 1:10 000, 1:5 000, 1:5 000 respectively. The high levels of specific antiserums of CA-derivatives were acquired.CONCLUSIONSConstruction of the tandem repetitive fusion peptide expression vector offers a new way to decrease the antibacterial toxicity to engineering bacteria and to raise the expression production.Comparing the activity of twoα-helicitys of CA,it has been found that the activity of CA19 is better than CA36. This suggests that the activity is correlated to the secondary structure.High levels of CA derivatives-specific antibodies can detect the CA which expresses in helerogeny expression systems.