ABSTRACT
During this study. we successfully expressed a codon-optimized gene for rotavirus VP6 protein intracellularly in two methylotrophic yeasts, Pichia pastoris and Hansenula polymorpha, during methanol induction. Expressions were performed in shake flasks and subsequently scaled-up to 1.3 L bioreactors. The yields obtained in the yeasts were compared with that observed in Escherichia coli. Despite producing the lowest biomass levels of all the expression systems in shake flasks, the highest VP6 concentration was obtained with E. coli. In shake flasks, P. pastoris yielded higher volumetric levels of VP6 than H. polymorpha, but specific production of VP6 was approximately similar in both yeasts. In the controlled environment of bioreactors, yeast strains attained typical high cell densities, but also increased VP6 production compared to all shake flask cultures. Unlike in shake flask expressions, H. polymorpha outperformed both P. pastoris as well as E. coli during bioreactor cultivation. VP6 production was in all three expression systems growth-associated. In contrast to yeast expressions, bacterial expressed VP6 protein was found to be insoluble upon analysis. This is the first report of VP6 expressed in methylotrophic yeast and holds the promise for the inexpensive production of VP6 as a possible vaccine candidate or drug delivery mechanism.
Subject(s)
Antigens, Viral/metabolism , Capsid Proteins/metabolism , Escherichia coli/metabolism , Pichia/metabolism , Recombinant Proteins/metabolism , Antigens, Viral/genetics , Capsid Proteins/genetics , Escherichia coli/genetics , Escherichia coli/growth & development , Gene Expression , Pichia/genetics , Pichia/growth & development , Recombinant Proteins/geneticsABSTRACT
Lactobacillus plantarum 24, isolated from marula fruit grows at pH 4.0 and tolerates acid levels and bile concentrations normally present in the human gastro-intestinal tract. Wistar rats that have been administered L. plantarum 24 showed no signs of discomfort or abnormal behavior. Tissue samples from the liver, spleen and intestine appeared normal. Furthermore, strain 24 harbors the genes encoding plantaricins A, F, and NC8α, a gene encoding immunity to plantaricin, and an ABC transporter similar in sequence to that reported for plantaricin G. At least one antimicrobial peptide within the size range of plantaricins A, F, and NC8α has been detected on a tricine-SDS-PAGE gel. Little is known about the microbial population in marula. This is the first report of a L. plantarum strain from marula fruit with bacteriocin genes and probiotic properties.
