Comparison of the extracellular full-length and truncated recombinant protein a production in Escherichia coli BL21 [DE3]

Protein A is a commercially important protein in biotechnological and medicinal applications. The great value of this protein and its applications in genetic and protein engineering and microbial researches as well as the growing use in biochemical industries, biotechnology, medicine and pharmacology, highlight the importance of the present study. In this survey the encoding genes of full-length and truncated forms of protein A were expressed in E. coli under an optimized expression condition. Optimization of the culture conditions resulted in an increase in expression and secretion of both forms of the protein, the pattern of expression and secretion levels for two forms was completely different. A minimum of 10-fold higher expression was observed for the truncated protein in comparison to that of the full-length recombinant form. Hydropathy plot of both forms of proteins showed that the missing domains in the truncated form contain groups of amino acids with high hydrophobicity score. Deletion of the terminal region could led to a higher expression level of the recombinant protein in E. coli. The function of these two proteins was studied using ELISA, which showed a higher activity for the truncated form for binding to IgG, compared to the full-length protein

Purification and characterization of 50 kDa extracellular metalloprotease from serratia sp. ZF03

Proteolytic enzymes have an important role in variety of physiological and pathological functions. They have been used in therapeutic and pharmaceutical applications. Characterizations of extracellular proteases from various strains of S. marcescens indicate that most strains produce a very similar major metalloprotease. This metalloprotease [serrapeptidase, serrapeptase] is an important pharmaceutical agent. Serrapeptase has been used in Asian and European countries for the treatment of inflammatory diseases, cardiovascular disorders, and bacterial infections. In the present study, purification and characterization of extracellular metalloprotease from Serratia sp. ZF03 for therapeutic purposes were reported. In this study the protease gene encoding a zinc-metalloprotease was isolated from the previously isolated red-pigmented Serratia sp. ZF03. The gene was sequenced and submitted to the GenBank. Proteolytic activity was detected by skim milk agar plate method and zymography. This fragment was found to encode an extracellular zincmetalloendopeptidase with a molecular weight of approximately 50 kDa. The metalloprotease was purified by ammonium sulfate precipitation and dialysis, and then characterized. The effects of various inhibitors and reagents on protease activity and its kinetic parameters were also determined. The nucleotide sequence demonstrated that deduced amino acid sequence has a higher identity with those of metalloprotease from serralysin family. Production of metalloprotease was highest at 48[th] h of cultivation. Optimum protease activity occurred at a temperature range of 50-55[degree sign]C and a pH range of 8.0-10. EDTA as a metal chelator, significantly inhibited protease activity. Zymography and inhibition assays showed that metalloprotease is the major secreted protease of Serratia sp. ZF03. The kinetic parameters, K[m] and V[m], were 0.00105 mg/ml and 0.0531 mM/min, respectively. Since the metalloprotease of this strain has strong proteolytic properties and good stability, it would be a suitable candidate to be used as an effective drug in the medicine and pharmaceutical industries

Effect of concomitant lycopene biosynthesis on CoQ[10] accumulation in transformed Escherichia coli strains

CoQ[10] and lycopene are isoprenoid compounds with nutraceutical and pharmaceutical benefits. In this study, the effect of concomitant lycopene biosynthesis on CoQ[10] accumulation in transformed Escherichia coli DH5 alpha was studied. A lycopene production pathway including geranyl diphosphate synthase [crtE], phytoene synthase [crtB], and phytoene desaturase [crtI] from Erwinia herbicola was constructed in two CoQ[10]-producing E. coli strains. E. coli Ba and E. coli Br containing dds orthologs encoding for decaprenyl diphosphate synthase [Dds], respectively from Agrobacterium tumefaciens and Rhodobacter sphaeroides were transformed by the lycopene pathway resulting in E. coli Ba-lyc and E. coli Br-lyc. The lycopene pathway in E. coli Br-lyc interestingly resulted in a significant increase in CoQ[10] production from 564 +/- 28 to 989 +/- 22 micro g /g DCW. To confirm that the improvement of CoQ[10] production in E. coli Br-lyc was due to lycopene biosynthesis and not just geranylgeranyl diphosphate formation in the lycopene pathway, crtE was only introduced into E. coli Ba and E. coli Br strains. Surprisingly, crtE expression had adverse effects on CoQ[10] production in both strains. The results shed light on the Dds-catalyzed reaction as a bottleneck controlled by precursors; and the efficiency of a parallel lycopene pathway to streamline the flow of metabolites