Orotidine-5'-monophosphate decarboxylase (OMPdecase) catalyzes the final step in pyrimidine biosynthesis, the conversion of orotidine-5'-monophosphate (OMP) to uridine-5'-monophosphate. The pyrF ...gene, encoding OMPdecase, was isolated from a chromosomal library of Pseudomonas aeruginosa PAO1 by screening for complementation of an Escherichia coli and a P. aeruginosa pyrF mutant. The nucleotide sequence of a 2510-bp chromosomal DNA fragment, complementing both strains, was determined (EMBL accession number X65613). On this a 696-bp open reading frame capable of encoding the 24 kDa OMPdecase was identified. Despite a generally good correspondence to other OMPdecase sequences, the P. aeruginosa gene was unique in that it did not constitute part of an operon. The pyrF gene was amplified by polymerase chain reaction, overexpressed in the pT7-7/E. coli BL21(DE3) system and purified to near electrophoretic homogeneity by anion exchange chromatography. Characterization of the purified enzyme revealed the following data, a Km value for OMP of 9.91 microM and an isoelectric point of 6.65. No major decrease in enzyme activity was observed in a pH range between 7.8 and 10.2. Gel electrophoresis under nondenaturing conditions suggested that the native form of OMPdecase is the dimer.
This chapter provides an introduction to the fascinating, useful, and sometimes dreaded world of protein purification and characterization. The goals of purification vary with the intended use of the ...purified protein. Protein purification can generally be divided into five broad stages, which do not all need to occur sequentially: (i) preparation of the source, (ii) gathering of all available information about the protein's properties, (iii) development of an assay, (iv) primary isolation, and (v) final purification. In recent years an autoinduction medium originally developed by Studier and now distributed by EMD Chemicals has gained an increased following in the protein expression community. Recovery and initial isolation steps separate the product from the majority of the water in the cultivation medium and from the majority of the host cell components. Recovery of both extracellular and intracellular proteins thus usually begins with the separation of the soluble culture medium from the insoluble cell pellet by centrifugation or filtration. Ion‐exchange chromatography is the most common high‐resolution method for preparative separation of proteins and is used in most protocols. Many eukaryotic (and a few prokaryotic) proteins contain sugars, which are sometimes essential to the protein's function. For instance, we now know that glycosylated proteins are involved in a wide variety of crucial molecular recognition events. Available methods include stains and blots for detection of glycosylation, deglycosylating enzymes, sugar‐specific lectins for blotting and separations, and nuclear magnetic resonance and mass spectrometry (MS) methods of characterizing individual side chains.
Serratia marcescens produces an endonuclease with extraordinarily high specific activity that is released into the surrounding medium. This enzyme has been the focus of studies on gene regulation, ...protein secretion, endonuclease action, and protein structure; it has also been found to have many applications in biotechnology. Here we briefly review these different facets of research regarding the
Serratia nuclease and summarize the current state of knowledge about this enzyme.
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