ABSTRACT
OBJECTIVE: To examine the potential of non-animal collagens as a new option for cosmetic applications. METHODS: Non-animal collagens from three species, Streptococcus pyogenes, Solibacter usitatus and Methylobacterium sp 4-46, have been expressed as recombinant proteins in Escherichia coli using a cold-shock, pCold, expression system. The proteins were purified using either metal affinity chromatography or a simple process based on precipitation and proteolytic digestion of impurities, which is suitable for large-scale production. Samples were examined using a range of analytical procedures. RESULTS: Analyses by gel electrophoresis and mass spectrometry were used to examine the purity and integrity of the products. Circular dichroism spectroscopy showed stabilities around 38°C, and calculated pI values were from 5.4 to 8.6. UV-visible light spectroscopy showed the clarity of collagen solutions. The collagens were soluble at low ionic strength between pH 5 and pH 8, but were less soluble under more acidic conditions. At lower pH, the insoluble material was well dispersed and did not form the fibrous associations and aggregates found with animal collagens. The materials were shown to be non-cytotoxic to cells in culture. CONCLUSIONS: These novel, non-animal collagens may be potential alternatives to animal collagens for inclusion in cosmetic formulations.
Subject(s)
Acidobacteria/chemistry , Collagen/chemistry , Cosmetics , Methylobacterium/chemistry , Streptococcus pyogenes/chemistryABSTRACT
The cancer-testis antigen, NY-ESO-1, has been engineered into a bacterial expression plasmid which incorporates a His6-tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15-litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY-ESO-1 tagged protein isolated as solubilised inclusion bodies. A three-step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY-ESO-1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.
Subject(s)
Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/isolation & purification , Cancer Vaccines/biosynthesis , Cancer Vaccines/isolation & purification , Membrane Proteins/biosynthesis , Membrane Proteins/isolation & purification , Protein Engineering/methods , Antigens, Neoplasm/genetics , Antigens, Neoplasm/therapeutic use , Australia , Cancer Vaccines/genetics , Drug Industry/standards , Guidelines as Topic , Humans , Membrane Proteins/genetics , Membrane Proteins/therapeutic use , Protein Engineering/standards , Quality Assurance, Health Care/standards , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Reference StandardsABSTRACT
Differing claims regarding the stability of the recombinant ethanologen E. coli KO11 are addressed here in batch and chemostat culture. In repeat batch culture, the organism was stable on glucose, mannose, xylose and galactose for at least three serial transfers, even in the absence of a selective antibiotic. Chemostat cultures on glucose were remarkably stable, but on mannose, xylose and a xylose/glucose mixture, they progressively lost their hyperethanologenicity. On xylose, the loss was irreversible, indicating genetic instability. The loss of hyperethanologenicity was accompanied by the production of high concentrations of acetic acid and by increasing biomass yields, suggesting that the higher ATP yield associated with acetate production may foster the growth of acetate-producing revertant strains. Plate counts on high chloramphenicol-containing medium, whether directly, or following preliminary growth on non-selective medium, were not a reliable indicator of high ethanologenicity during chemostat culture. In batch culture, the organism appeared to retain its promise for ethanol production from lignocellulosics and concerns that antibiotics may need to be included in all media appear unfounded.
