ABSTRACT
Extracellular lipase Lip2 from Yarrowia lipolytica is a promising biocatalyst with unusual structural features, as indicated by X-ray crystallography. These features comprise a mobile domain called the lid that controls access to the catalytic site. Conformational rearrangements of the lid have been suggested to regulate lipase enzymatic activities. We used nuclear magnetic resonance to investigate the dynamics of Lip2 by exploring four expression systems, Escherichia coli, cell-free, Pichia pastoris and Y. lipolytica to produce uniformly labelled enzyme. The expression of Lip2 was assessed by determining its specific activity and measuring (15)N-(1)H HSQC spectra. Y. lipolytica turned out to be the most efficient expression system. Here, we report the first use of Y. lipolytica as an expression host for the production of uniform stable isotopic labelled protein for further structural and dynamics studies using NMR.
Subject(s)
Fungal Proteins/biosynthesis , Gene Expression/genetics , Isotope Labeling/methods , Lipase/biosynthesis , Yarrowia/enzymology , Yarrowia/metabolism , Catalytic Domain , Cell-Free System/metabolism , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Lipase/chemistry , Lipase/genetics , Nuclear Magnetic Resonance, Biomolecular , Pichia/genetics , Pichia/metabolism , Yarrowia/geneticsABSTRACT
The spatial-temporal distribution of the mRNAs for type IX and type XI collagens were compared to that of type II collagen mRNA in the tibial epiphyseal plate cartilage of normal growing rats. The mRNAs were detected by in situ hybridization with radio-labelled specific probes and visualized by radioautography. The areas covered by the resulting silver grains were quantified by computer assisted image analysis. The areas in chondrocytes of each zone of the epiphyseal plate cartilage, which correspond to the stages of chondrocyte development and function were determined. Types II, IX and XI mRNAs were present to some extent in chondrocytes of all zones. The distributions of type II and type IX collagen mRNAs were similar with the highest concentrations in the proliferative zone, and the lowest in the resting and calcifying zones chondrocytes. In contrast, type XI collagen mRNA had a different distribution, with the lowest concentration in the resting zone chondrocytes and a significant decrease in the calcifying zone chondrocytes. These patterns correlates with the changes in chondrocyte function, and may reflect the roles of the type IX and type XI collagens. The data show that computer assisted image analysis of in situ hybridization radioautographic images is a precise, rapid tool for analysing differences in gene expression.
