Effect of Twine-arginine Translocation-signaling Fusion System and Chaperones Co-expression on Secretory Expression of Somatropin.

BACKGROUND: Twine-arginine translocation (TAT) system is one of the exporting systems in Escherichia coli which could transport fully/semi-correctly folded proteins outside the reductive cytoplasmic space. In combination with co-expression with a chaperone system, the correctly folded proteins could be transported to oxidative periplasmic space and culture media to pass the main limitations in E. coli expression system such as misfolding and inclusion body formation. MATERIALS AND METHODS: To study the effectiveness of signaling sequences and chaperone co-expression on the translocation of expressed protein, somatropin was selected as the target. Two common signal sequences in TAT system (TorA and SufI) were added at the N-terminal of somatropin and the cassettes were co-expressed in E. coli BL21 (DE3) by a chaperone team including DnaK/J-GrpeE. RESULTS: The expression pattern studies including Western blotting and sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that somatropin is expressed in two cassettes. However, the pattern was different for two signaling sequences. CONCLUSION: The results confirmed that the approach of using TAT-signaling sequences and co-expression with the chaperone team could enhance translocation of protein to periplasmic space and culture media compared to control groups. Western blotting results showed that the signal sequence TorA could transport more expressed proteins to the periplasmic space and culture media in comparison with SufI. However, there was a considerable amount of human growth hormone in the cytoplasm which could not be transported outside the cytoplasmic space.

Twin arginine translocation system in secretory expression of recombinant human growth hormone.

Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.

Microbial quality survey of sunscreen products in Iranian market.

BACKGROUND: Microbial contamination of cosmetic products is very crucial because of their daily use and direct contact with the skin. These products are at high risk for microbial contamination from various sources such as environment, consumer's hands, body sweat and during the time of manufacturing. Therefore, this study aimed to investigate the microbial quality of sunscreens products, manufactured in or imported to or formulated in local pharmacies in Iran. MATERIALS AND METHODS: The microbial quality were determined in three different levels; the intact product (at the time of purchase) and after three and after six months of opening it. Total Aerobic Viable Count (TAVC) and the presence of coliforms, Pseudomonas aeruginosa, Staphylococcus aureus, molds, and yeasts were studied. RESULTS: At the time of purchase, 40, 73.3 and 43.3 percentage of Iranian made, imported and pharmacy formulated sunscreens were contaminated with at least one of the objectionable microorganisms, respectively. After three months of opening it, 36.6, 70 and 46.6 percentage of Iranian made, imported and pharmacy formulated sunscreens were contaminated with at least one of the objectionable microorganisms, respectively. The percentages of contaminated samples were 36.6, 70 and 50 after six months of opening for Iranian made, imported and pharmacy formulated sunscreens, respectively. CONCLUSION: Microbial contamination of these sunscreens products is a potential health risk for consumers. It seems that it is necessary to inspect and monitor the products during the manufacturing and shelf life period. It is highly recommended to control and regulate cosmetic products by health organizations to ensure the quality and safety of this kind of products.

Isolation and characterization of novel thermophilic lipase-secreting bacteria

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

Isolation and characterization of novel thermophilic lipase-secreting bacteria.

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.