PxCECA1

To produce recombinant Buthus martensii Karsch insect toxin (BmK IT), BmK IT cDNA which fused a hexahistidine sequence at the C-terminus by PCR was inserted into pTWIN1 expression vector fused in frame with an upstream Ssp DnaB intein gene. The expression plasmid was transformed into E. coli BL21 (DE3) strain and protein expression was induced by IPTG. The CBD-Intein-BmK IT(his6) fusion protein was purified from cell lysates using Ni-NTA resin affinity chromatography. The intein was removed from fusion protein by on-column intein-mediated cleavage. BmK IT(his6) was purified through Superdex 75 gel chromatography to more than 95% homogeneity. The purified protein has both correct secondary structure and insecticidal activity.

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in host defense. It targets the beta (1-4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making lysozyme highly active against Gram-positive bacteria. However, lysozyme alone is inactive against Gram-negative bacteria because it cannot reach the peptidoglycan layer. Cecropins are cationic molecules with a wide range of antimicrobial activities. The main target for these peptides is the cytoplasmic membrane. We resume that cecopin may disrupt the outer membrane, giving the enzyme access to the peptidoglycan in cell wall. So in the present study, novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was designed. The DNA sequence encoding recombination fusion protein Mdc-hly was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). The protein was expressed as a His-tagged fusion protein, and the Mdc-hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. Antimicrobial activity assays showed that the recombinant fusion protein Mdc-hly has improved in vitro antimicrobial activity and action spectrum compared to Mdc and hly. Mdc-hly may have important potential application as a future safely administered human drug and food additive.

Insect cecropins are small basic polypeptides synthesized in fat body and hemocytes in response to bacterial infections or hypodermic injuries. To explore a new approach for high expression of soluble cecropin in Escherichia coli cells, we fused the sequence encoding Musca domestica mature cecropin (named Mdmcec) in-frame to thioredoxin (TRX) gene to construct an expression vector pTRX-6His-Mdmcec. An enterokinase cleavage site was introduced between the 6xHis-tag and Mdmcec to facilitate final release of the recombinant Mdmcec. The fusion protein TRX-6His-Mdmcec was purified successfully by HisTrap HP affinity column and a high yield of 48.0mg purified fusion protein was obtained from 1L culture. Recombinant Mdmcec was readily obtained by enterokinase cleavage of the fusion protein followed by HPLC chromatography, and 11.2mg pure active recombinant Mdmcec was obtained from 1L E. coli culture. The molecular mass of recombinant Mdmcec determined by electrospray ionization-mass spectrometry (ESI-MS) is identical to that of native cecropin. Analysis of recombinant Mdmcec by circular dichroism (CD) indicated that recombinant Mdmcec contained predominantly alpha-helix with some random coil. Antimicrobial activity assays demonstrated that recombinant Mdmcec had a broad spectrum of activity against fungi, Gram-positive and negative bacteria. The procedure described in this study will provide a reliable and simple method for production of different cationic peptides for biological studies.