شاهین حدادیان

 Endotoxin or lipopolysaccharide (LPS) is one of the components of the wall of gram-negative bacteria, which, when in contact with blood, stimulates the immune system and causes fever and even serious adverse effects or death. Removing endotoxin is one of the most daunting challenges presented to the purification process in the production of recombinant biological drugs. Bacterial endotoxin (LPS) is resistant to heat and passes through sterilizing filters, and due to its dangerous side effects in living organisms, part of the target protein purification process is always related to removing this disturbing factor from the product.

The affinity matrix S3E3-S-Sepharose, which was produced by immobilizing the improved variant of antimicrobial peptide S3 called S3E3 peptide on Sepharose chromatography resin, was used to remove endotoxin from active pharmaceutical ingredient (API) of streptokinase, and the performance of this matrix was compared with the performance of a disposable commercial matrix (containing S3 peptide as its ligand) and with ion exchange chromatography resin.

The statistical analysis of the results revealed that compared to commercial S3 matrices and ion exchange chromatography, the S3E3-S-Sepharose matrix had higher protein recovery (84.07% compared to 81.50 and 75.31%, respectively) and higher streptokinase biological activity recovery (81.95% vs. 27 76.76 and 61.54%, respectively).

As evidenced by the obtained results, the S3E3-S-Sepharose matrix seems to be a suitable candidate for use in the purification processes of Streptokinase API and other recombinant biopharmaceuticals.

Antimicrobial peptides (AMPs) are compounds with antimicrobial properties that are studied widely due to the development of resistance of pathogenic bacteria to antibiotics. In the present study, the toxicity and antimicrobial effects of two natural monomeric peptides (S3 and S∆3) were compared with S3-S∆3 hybrids and S3 tetramers.

Protein hybrids (S∆3S3-2mer-GS) S3-S∆3 and tetramer protein S3 (S3-4mer-GS) were expressed in E. coli. BL21 (DE3). Following that, the presence of mutant peptides was confirmed, and their antimicrobial activity was compared and evaluated with S3 and S∆3 monomers. Finally, the toxicity of tetramer and hybrid made on the MDA-MB-231 cell line was evaluated and compared.

The toxicity of the hybrid was slightly increased, compared to the tetramer for eukaryotic cells; however, this increase was negligible at the active concentration of this protein. Cell survival for hybrids was lower for S3 and SΔ3; nonetheless, cell survival for each sequence decreased with increasing time. Furthermore, the inhibition of hybrid microbial growth was improved and compared with tetramer and S3-SΔ3. It was found that an increase in the positive charge of the hybrid protein did not have a toxic effect on the host bacteria.

Due to the appropriate expression and increased antimicrobial activity and negligible cytotoxicity, the hybrid peptide S3-S∆3 and tetramer S3 can be considered an effective production strategy to obtain AMPs.

The study of the effects of synthetic peptides with antibacterial properties can provide more effective antibiotics. This study designed, expressed, and investigated the Sushi 3 tetramer peptide. Subsequently, it was compared in terms of changing antibacterial properties with another Sushi3 tetramer peptide the aspartic acid and proline amino acids of which were replaced with glycine and serine amino acids.

First, the mentioned Sushi3 tetramer peptide sequences were designed, constructed, and named Mer1 and Mer 2, respectively, and cloned separately into plasmid pET-26b (+) and finally transferred to E.coli BL21 host (DE3). After the expression of the peptides, the presence of peptides was confirmed by SDS-PAGE and Western blotting. Afterward, the antimicrobial activity of Mer1 and Mer 2 was evaluated and compared. Finally, the toxicity of the two tetramers made on the MDA-MB-231 cell line was evaluated and compared.

Mer1 and Mer 2 had similar protein expression, and the toxic effect of both peptides on the cell line was not significantly different. however, Mer 2 had more effective antimicrobial effects than Mer1 at the same concentrations.

Evaluation of the effect of amino acid replacement with less negatively charge on increasing the antimicrobial activity of peptides is a suitable strategy. The above results increase the possibility of designing and producing antimicrobial peptides against antibiotic-resistant strains as the next generation of antibiotics.

Vibrio cholera is a gram-negative bacterium that causes cholera which is an acute diarrheal illness. Cholera outbreak mostly happens in hot seasons when the bacteria reproduce rapidly in water contaminated with feces. Severe diarrhea and mortality have been the prevalent symptoms of cholera which affect the hygienic status and economic features of humans during centuries. There is a challenge currently to plan and propose a quick and reliable method to detect vibrio cholera which has been one of the fast growing and contagious disease in this decade. New Zealand white rabbits were immunized with vibrio cholera Ogawa and Inaba whole cells. The obtained gamma globulin from the whole serum was purified through ammonium sulfate precipitation method and then concentrated in this study. Subsequently, the purity of these antigens increased with the use of mixed mode-chromatography in the next step. The purified antibodies were connected to staphylococcus aureus cowan 1 NCTC-8325 whole cells. Rectal swab samples were inoculated into an enriched Alkaline Peptone Water medium (pH 8.6) incubated for 5 h at 37 °C. One drop from each sample was mixed with one drop of vibrio cholera co-agglutination complex. Subsequently, the results were read in 2-3 min. The obtained results showed 97%, 99%, and 98% of sensitivity, specificity, and accuracy from the proposed assay, respectively, compared to the standard culture methods. This proposed assay is considered as a rapid and reliable method to evaluate endemic or epidemic transmission of fatal illnesses. The proposed co-agglutination test is a simple, rapid, and reliable method to detect Vibrio cholera in fecal specimens. This method can be utilized to detect and control severe diarrhea which results from vibrio leading to the prevention of mortality, especially in areas with minimal laboratory facilities.