Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. This study attempted to evaluate the five host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHuffle in terms of arginine deiminase (ADI) production and enzyme activity. Arginine deiminase (ADI) was chosen a bacterial enzyme which degrades L-arginine. It is effective in treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC) which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through CaCl 2 method. It was then transformed with the construct of pET3a-ADI using heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

Background: Pharyngeal carriers are the source and transitional vectors of invasive diseases. Attachment is the first step in pathogenicity. Strains of Streptococcus as normal flora can cause diseases in certain circumstances. Adhesion proteins of these bacteria play a fundamental role in the attachment and colonization.

Background: Arginine deiminase is a bacterial enzyme which degrades L-arginine. Some human cancers such as hepatocellular carcinoma (HCC) and melanoma are auxotrophic for arginine. Therefore, PEGylated arginine deiminase (ADI-PEG20) is a good anticancer candidate with antitumor effect. It causes local depletion of L-arginine and growth inhibition in arginine-auxotrophic tumor cells. The FDA and EMA have granted orphan status to this drug. Objective: Due to its increasing attention, we aimed to evaluate and compare 30 arginine deiminase proteins from different bacterial species through in silico analysis. Methods: The exploited analyses included investigation of physicochemical properties, multiple sequence alignment (MSA), motif, superfamily, phylogenetic and 3D comparative analyses of arginine deiminase proteins thorough various bioinformatics tools. Results: The most abundant amino acid in the arginine deiminase proteins is leucine (10.13%) while the least amino acid ratio was cysteine (0.98%). Multiple sequence alignment showed 47 conserved patterns between 30 arginine deiminase amino acid sequences. The results of sequence homology among 30 different groups of arginine deiminase enzymes revealed that all the studied sequences located in amidinotransferase superfamily. Based upon the phylogenetic analysis two major clusters were identified. Considering the results of various in silico studies; we selected the five best candidates for further investigations. The 3D structures of the best five arginine deiminase proteins were generated by I-TASSER server and PyMOL. The RAMPAGE analysis revealed that 81.4%-91.4%, of the selected sequences were located in the favored region of arginine deiminase proteins. Conclusion: The results of this study shed light on the basic physicochemical properties of thirty major arginine deiminase sequences. The obtained data could be employed for further in vivo and clinical studies and also developing the related therapeutic enzymes.

Aims: This study attempted to evaluate the five host strains, including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE, and SHuffle, in terms of arginine deiminase (ADI) production and enzyme activity. Background: Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors, and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. Various E. coli cells have been examined in different patent applications. Method: ADI was chosen as a bacterial enzyme that degrades L-arginine. It is effective in the treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC), which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through the CaCl2 method. It was then transformed with the construct of pET3a-ADI using the heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for the expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. Result: All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein, followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. Conclusion: There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.