Production of Soluble and Functional Anti-TNF-α Fab' Fragment in Cytoplasm of E. coli: Investigating the Effect of Process Conditions on Cellular Biomass and Protein Yield Using Response Surface Methodology.

With the increasing dominance of monoclonal antibodies (mAbs) in the biopharmaceutical industry and smaller antibody fragments bringing notable advantages over full-length antibodies, it is of considerable significance to choose the most suitable production system. Although mammalian expression system has been the preferred choice in recent years for mAbs production, E. coli could be the favorable host for non-glycosylated small antibody fragments due to the emergence of new engineered E. coli strains capable of forming disulfide-bonds in their cytoplasm.In this study, non-glycosylated anti-TNF-α Fab' moiety of Certolizumab pegol, produced by periplasmic expression in E. coli in previous studies, was produced in the cytoplasm of E. coli SHuffle strain. The results indicated that it is biologically functional by testing the antigen-binding activity via indirect ELISA and inhibition of TNF-α induced cytotoxicity using MTT test. Major factors affecting protein production and, optimized culture conditions were examined by analyzing growth characteristics and patterns of expression in 24 h of post-induction cultivation and, optimization of culture conditions by response surface methodology considering temperature, time of induction and concentration of inducer in small (tube) and shake-flask scale. Based on the results, temperature had the most significant influence on functional protein yield while exerting different impacts in small and shake-flask scales, which indicated that cultivation volume is also an important factor that should be taken into account in optimization process. Furthermore, richness of medium and slower cellular growth rate improved specific cellular yield of functional protein by having a positive effect on the solubility of Fab' antibody.

Improvement of Certolizumab Fab' properties by PASylation technology.

Certolizumab pegol is a Fab' antibody fragment for treatment of rheumatoid arthritis and Crohn's disease which is conjugated to a 40 kDa PEG molecule in order to increase the protein half-life. PEGylation may have disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity of the protein. To overcome these problems, PASylation has been developed as a new approach. The nucleotide sequence encoding 400 amino acid PAS residues was genetically fused to the corresponding nucleotide sequences of both chains of certolizumab. Then, the bioactivity as well as physicochemical and pharmacokinetic properties of the recombinant PASylated expressed protein was assayed. Circular dichroism spectroscopy demonstrated that the random coil structure of PAS sequences did not change the secondary structure of the PASylated Fab' molecule. It was observed that PASylation influenced the properties of the Fab' molecule by which the hydrodynamic radius and neutralization activity were increased. Also, the antigen binding and binding kinetic parameters improved in comparison to the PEGylated Fab' antibody. Pharmacokinetic studies also showed prolonged terminal half-life and improved pharmacokinetic parameters in PASylated recombinant protein in comparison to the PEGylated and Fab' control molecules. The results reconfirmed the efficiency of PASylation approach as a potential alternative method in increasing the half-life of pharmaceutical proteins.

Isolation of Enteropathogenic Escherichia coli from lettuce samples in Tehran.

AIM: The purpose of this study was to find the isolation rate of enteropathogenic Escherichia coli (EPEC) from lettuce samples collected in Tehran. BACKGROUND: During the last decade, the prevalence of infectious diarrheal diseases due to consumption of contaminated food especially raw vegetable has been increasingly reported. Enteropathogenic Escherichia coli strains are an important group of diarrheagenic E. coli that can cause infant diarrhea especially in the developing world. MATERIAL AND METHODS: One hundred lettuce samples collected in Tehran were transported to the laboratory, homogenized by a stomacher in EC broth containing cefixime, and cultured on MacConkey agar plates. Bacterial DNA was extracted by boiling method and PCR was performed using three pairs of primers targeting stx 1, stx 2 and eaeA genes. RESULTS: Screening of 100 lettuce samples by PCR showed four samples were positive for the presence of EPEC. CONCLUSION: This study suggests contamination of the lettuce by the EPEC and its possible role as the source of infection in this region.

Shiga toxin-producing Escherichia coli isolated from lettuce samples in tehran, iran.

BACKGROUND: During the last decade, the prevalence of foodborne diseases due to contaminated food as well as the outbreaks of diseases due to Shiga toxin-producing Escherichia coli (STEC) strains has increased. OBJECTIVES: The aim of this study was to evaluate the prevalence and antibiotic resistance pattern of STEC strains in lettuce samples. Since lettuce is used as a raw vegetable in salads, the rates of infections caused by this vegetable are high. MATERIALS AND METHODS: A total of 100 samples collected from Tehran, Iran, were transported to the laboratory, homogenized by a stomacher in E. coli broth containing cefixime, and cultured on MacConkey agar medium. Their DNA was extracted by boiling method and polymerase chain reaction (PCR) was performed, using five primers targeting the stx1, stx2, fliCh7, rbfO157, and eaeA genes. Susceptibility testing against ampicillin, imipenem, cephalosporin, tetracycline, aminoglycosides, chloramphenicol and quinolones was performed using disk diffusion method. RESULTS: Eight samples were positive for presence of STEC strains, three contained stx1, five contained stx2, and one sample was positive for presence of both rbfO157 and fliCh7. They were susceptible to all the antibiotics except for ampicillin and tetracycline. CONCLUSIONS: This study indicated the contamination of lettuce by STEC strains and its possible role as the source of infection. Resistance to both tetracycline and ampicillin may be considered as an emergency alarm for a multidrug resistance of STEC strains.