Overexpression, overproduction, purification, and characterization of rhGH in Escherichia coli.

Overexpression of insoluble human growth hormone (hGH) in cytoplasm was achieved by E. coli Rosetta-gami B(DE3) [pET21a (+)-hGH]). For overexpression of hGH, effects of eight factors including temperature, type and concentration of carbon source, IPTG and MgSO4 , buffering capacity, induction time, yeast extract/peptone ratio on rhGH production were studied by Plackett-Burman screening. Maximum production of rhGH was 0.681 g/L, and results of statistical analysis showed that induction temperature and glucose have the greatest effect and the presence of MgSO4 increases rhGH expression and reduces biomass concentration. So, the effect of ethanol and MgSO4 concentrations on the rhGH production was examined according to the central composite experimental design. The ANOVA of the results showed rhGH production increases to 1.128 g/L in 4 g/L MgSO4 and 1% ethanol. Then, the impact of glucose concentration and induction time on the rhGH production was evaluated in two levels in the fermenter by Taguchi statistical method. Under optimum conditions, OD600nm 4 and 10 g/L glucose crude rhGH concentration 4.17 g/L was obtained, which is one of the highest value ever reported. Finally, rhGH was purified using the biophysical and biochemical techniques comprising circular dichroism, fluorescent spectroscopy, and dynamic light scattering, and it was confirmed that the produced protein is comparable to the commercial standard sample.

Increased cellulose production by heterologous expression of bcsA and B genes from Gluconacetobacterxylinus in E. coli Nissle 1917.

Based on cellulose biosynthesis pathway of Gluconacetobacterxylinus BPR2001 and E. coli Nissle 1917, bcsA and bcsB genes have been selected and bioinformatics studies done to the analyses of nucleotide and amino acid sequence alignment, stability of RNA, protein, and promotor power. We amplify and clone bcsA, bcsB, and bcsAB genes of G. xylinus BPR2001 in Escherichiacoli Nissle 1917 under the inducible tac promoter. Our results of bioinformatics predictions demonstrate similar active site and three-dimensional structure of BcsA and BcsB proteins in two different bacteria. In addition, our data reveal that BcsA and BcsB proteins of E. coli have weaker promotor power, RNA secondary structure, and protein stability than that of the same proteins in G. xylinus. Some of the reasons of BcsAB protein selection from G. xylinus and its heterologous expression in E. coli is the noted points. Production of the related proteins visualized using SDS-PAGE. We find out that Congo red absorbance at 490 nm has no significant difference in wild-type strain (E. coli Nissle 1917) compared to recombinants bcsA+ or bcsB+, but recombinant bcsAB+ could produce more cellulose than that of the wild-type strain. Furthermore, the measurement of cellulose dry weights of all samples confirms bacterial cellulose production enhancement in recombinant bcsAB+ (1.94 g l-1). The FTIR analysis reveals that the crystallinity indices do not change significantly after over expressing each of genes.

In silico and experimental improvement of bacteriorhodopsin production in Halobacterium salinarum R1 by increasing DNA-binding affinity of Bat through Q661R/Q665R substitutions in HTH motif.

DNA-binding motif of bacterioopsin activator (Bat) protein is a Helix-Turn-Helix motif, which binds to bop promoter and induces bacterioopsin (Bop) expression under light and low oxygen tension. Bacterioopsin is linked to retinal to produce bacteriorhodopsin (BR), which in turn supplies energy source in Halobacterium salinarum. In this study, effect of Bat HTH motif-promoter DNA interaction on bacterioopsin (Bop) expression was investigated using in silico and experimental approaches. Molecular docking showed that the most stable DNA-protein complex was generated by Q661R/Q665R mutant. Based on the in silico analysis, HTH motif was mutated using site-directed mutagenesis and Hbt. salinarum recombinant strains were developed by introduction of mutant bat genes. Double positively charged amino acid substitutions (Q661R/Q665R) in second helix of HTH motif increased whereas deletion of this region decreased BR production. However, other single substitutions (Q665R and Q661H) did not change BR production. These findings represent key role of HTH motif stability for DNA binding and regulation of bacterioopsin (Bop) expression and bacteriorhodopsin (BR) production independent of environmental condition.

Enhancement of crystallinity of cellulose produced by Escherichia coli through heterologous expression of bcsD gene from Gluconacetobacter xylinus.

OBJECTIVES: To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001. RESULTS: The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD. CONCLUSION: The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.