Expression and secretion of cyan fluorescent protein [CFP] in B. subtilis using the chitinase promoter from bacillus pumilus SG2

Background: Improved cyan fluorescent protein [ICFP] is a monochromic, green fluorescent protein [GFP] derivative produced by Aequorea macrodactyla in a process similar to GFP. This protein has strong absorption spectra at wavelengths 426-446 nm. ICFP can be used in cell, organelle or intracellular protein labeling, investigating the protein-protein interactions as well as assessing the promoter activities

Methods: In our previous study, the promoters of two chitinases [ChiS and ChiL] from Bacillus pumilus SG2 were assessed in B. subtilis and their regulatory elements were characterized. In the present study, icfp was cloned downstream of several truncated promoters obtained in the former study, and ICFP expression was evaluated in B. subtilis

Results: Extracellular expression and secretion of ICFP were analyzed under the control of different truncated versions of ChiSL promoters grown on different media. Results from SDS-PAGE and fluorimetric analyses showed that there were different expression rates of CFP; however, the UPChi-ICFP3 construct exhibited a higher level of expression and secretion in the culture medium

Conclusion: Our presented results revealed that inserting this truncated form of Chi promoter upstream of the ICFP, as a reporter gene, in B. subtilis led to an approximately ten fold increase in ICFP expression

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

alternative bacterial expression system using bacillus pumilus SG2 chitinase promoter

Background: Chitin is an abundant natural polysaccharide found in fungi, algae, and exoskeleton of insects. Several bacterial species are capable of utilizing chitin as their carbon source. These bacteria produce chitinases for degradation of chitin into N-acetyl-D-glucosamine. So far, regulation of the chitinase encoding genes has been studied in different bacterial species. Among Bacillus species, B. pumilus strain SG2 encodes two chitinases, ChiS and ChiL. The promoter region of chiSL genes [PchiS] is mainly regulated by the general carbon catabolite repression [CCR] system in B. subtilis due to the presence of a catabolite responsive element [cre]

Objectives: Use of P[chiS] in constructing an inducible expression system in B. subtilis was investigated

Materials and Methods: In the first step, complete and shortened versions of P[chiS] were inserted upstream of the lacZ on a pBS72/pUC18 shuttle plasmid. The beta-galactosidase activity of B. subtilis carrying one of the relevant plasmids was measured in the presence of different carbon sources

Results: An expression system based on the chitinase promoter of B. pumilus SG2 was established. Modification of PchiS and the culture medium resulted in production of beta-galactosidase in B. subtilis up to 1,800 Miller unit [MU] activity

Conclusions: The chitinase promoter developed in this study, has potential to be used in an expression vector that could be induced by chitin. In addition, compared to the other inducers like IPTG and lactose, chitin is definitely cheaper and more available as an inducer