Production of d-Tagatose by Whole-Cell Conversion of Recombinant Bacillus subtilis in the Absence of Antibiotics

d-tagatose is a popular functional monosaccharide produced from lactose by β-galactosidase and arabinose isomerase. In this study, two d-alanine-deficient heterologous gene expression systems were constructed, B. subtilis 168 D1 and B. subtilis 168 D2, using overlapping extension PCR and the CRE/loxP system. The lacZ gene for β-galactosidase was integrated into a specific locus of the chassis B. subtilis 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene alrA attached to it was constructed and used to assemble recombinant plasmids overexpressing β-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene araA of E. coli transform-competent B. subtilis 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout B. subtilis 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L d-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. B. subtilis 168 D1/pMA5-alrA-araA-lacZ is capable of single-cell one-step production of d-tagatose. This study provides a new approach to the production of functional sugars.

Production and purification of constructed recombinant hirudin in BL21(DE3) strain

Background/Objective: Hirudin, an extract from the leech, has powerful antithrombin activity affecting the blood coagulation pathway, it is the most potent natural inhibitor of thrombin, it binds thrombin with high affinity, so, the aim of this study was to build hirudin gene by overlapping extension PCR then cloning and expression in BL21(DE3) strain.Methods: Hirudin gene constructed with four modified primers then the final product amplified by two primers named as A, B by using overlapping extension PCR, for gene expression, BL21(DE3) strain was used under the control of T7 promoter in pET-16b vector and for hirudin production, LB broth medium was used as fermentation medium. Hirudin protein purified at first by (Immobilized metal affinity chromatography) IMAC, then this protein was dialysis and treated with factor Xa to eliminate His-tag. Then hirudin purified by DEAE sepharose and SP sepharose column, the concentration of protein determined by ELISA, furthermore the activity evaluated by thrombin titration and activated partial thromboplastin time (APPT) test.Results: Hirudin gene constructed in two round PCR first round produced two products (product1 117 bp while product 2,114 bp) the second-round PCR gave the final product 213 bp. The resulted band from gel electrophoresis for constructed vector pET-16b-HirudinS was (5,901 bp). The analysis on 15% SDS-PAGE for the SP sepharose column illustrated the hirudinS protein band with size about ~10.8. Concentration of produced hirudin within its solution reached to 1.75 ng, thrombin titration method showed that the hirudin protein required 300 µl from thrombin to clot, also, APPT test showed that hirudin elongated clotting time to 7min in comparison with 6min for aspirin and the statistical analysis results for APPT test illustrated that there was no significant difference between hirudin and aspirin.Conclusion: This study approved that overlapping extension PCR is a good strategy for building hirudin gene and it’s successfully expressed in BL21(DE3) strain.

Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of aPlasmodiumRegion I-Plus Peptide

Interferon alpha (IFNα) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFNαis the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFNαmay account for its lowin vivobiological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFNα2b with aPlasmodiumregion I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression inE. coliBL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentialityin vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent.

Extracellular Expression of a Functional Recombinant Ganoderma lucidium Immunomodulatory Protein by Bacillus subtilis and Lactococcus lactis

ABSTRACT Bacillus subtilis and Lactococcus lactis are ideal hosts for the production of extracellular heterologous proteins of major commercial importance. A recombinant gene for the novel Ganoderma lucidium immunomodulatory protein LZ-8, recombinant LZ-8, was designed encoding the same amino acid sequence but using the preferred codons for both strains and was synthesized by overlapping extension PCR. Using the signal peptide (SP) from subtilisin YaB (SPYaB), recombinant LZ-8 was expressed extracellularly in Bacillus subtilis and Lactococcus lactis. In the absence of SPYaB, recombinant LZ-8 was expressed extracellularly in B. subtilis, but not in L. lactis. The three expressed recombinant LZ-8s showed different capacities for modulating the production of Th1 and Th2 cytokines by peripheral blood mononuclear cells and of tumor necrosis factor alpha by a macrophage cell line.