Computational design of N-linked glycans for high throughput epitope profiling.

Efficient identification of epitopes is crucial for drug discovery and design as it enables the selection of optimal epitopes, expansion of lead antibody diversity, and verification of binding interface. Although high-resolution low throughput methods like x-ray crystallography can determine epitopes or protein-protein interactions accurately, they are time-consuming and can only be applied to a limited number of complexes. To overcome these limitations, we have developed a rapid computational method that incorporates N-linked glycans to mask epitopes or protein interaction surfaces, thereby providing a mapping of these regions. Using human coagulation factor IXa (fIXa) as a model system, we computationally screened 158 positions and expressed 98 variants to test experimentally for epitope mapping. We were able to delineate epitopes rapidly and reliably through the insertion of N-linked glycans that efficiently disrupted binding in a site-selective manner. To validate the efficacy of our method, we conducted ELISA experiments and high-throughput yeast surface display assays. Furthermore, x-ray crystallography was employed to verify the results, thereby recapitulating through the method of N-linked glycans a coarse-grained mapping of the epitope.

Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3).

The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni(2+)-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC(50) of 3.6 and 1.9 microM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides.

Design and characterization of novel hybrid peptides from LFB15(W4,10), HP(2-20), and cecropin A based on structure parameters by computer-aided method.

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. The pivotal assets of the antimicrobial peptide include potential for rapid bactericidal activity and low propensity for resistance. The four new antimicrobial hybrid peptides were designed based on peptides LFB15(W4,10), HP(2-20), and cecropin A according to the structure-activity relationship of the amphipathic and cationic antimicrobial peptides. Their structural parameters were accessed by bioinformatics tools, and then two hybrids with the most potential candidates were synthesized. The hybrid peptide LH28 caused an increase in antibiotic activity (MIC(50)=1.56-3.13 microM) against given bacterial strains and did not cause obvious hemolysis of rabbit erythrocytes at concentration of 3.13 microM with effective antimicrobial activity. The results demonstrate that evaluating the structural parameters could be useful for designing novel antimicrobial peptides.