David Landry

SINGLE-COLUMN PURIFICATION OF FREE RECOMBINANT PROTEINS USING A SELF-CLEAVABLE AFFINITY TAG DERIVED FROM A PROTEIN SPLICING ELEMENT

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

Novel endo-α-N-acetylgalactosaminidases with broader substrate specificity

In an effort to identify novel endo-α-N-acetylgalact- osaminidases (endo-α-GalNAcases), four potential genes were cloned. Three of the expressed proteins EngEF from Enterococcus faecalis, EngPA from Propionibacterium acnes, and EngCP from Clostridium perfringens were purified and characterized. Their substrate specificity was investigated and compared to the commercially available endo-α-GalNAcases from Streptococcus pneumoniae (EngSP) and Alcaligenes sp. (EngAL). All enzymes were incubated with various synthetic substrates, and natural glycoproteins and the released sugars were detected by colorimetric assay and thin layer chromatography analysis. The Core 1 disaccharide Galβ1,3GalNAcα1pNP was the most rapidly hydrolyzed substrate by all enzymes tested. EngEF exhibited the highest kcat for this substrate. EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAcβ1,3GalNAcα1pNP. This is the first report of endo-α-GalNAcases EngEF and EngPA acting on Core 3 in addition to Core 1 O-glycans. Interestingly, there were no significant differences in transglycosylation activities when Galβ1,3GalNAcα1pNP or GlcNAcβ1,3GalNAcα1pNP was incubated with various 1-alkanols in the presence of the endo-α-GalNAcases tested in this work.

M · FokI methylates adenine in both strands of its asymmetric recognition sequence

M.FokI, a type-IIS modification enzyme from Flavobacterium okeanokoites, was purified, and its activity was characterized in vitro. The enzyme was found to be a DNA-adenine methyltransferase and to methylate both strands of the asymmetric FokI recognition sequence: (formula; see text) M.FokI does not methylate single-stranded DNA, nor does it methylate double-stranded DNA at sequences other than FokI sites.

Characterization of type II DNA-methyltransferases.

Publisher Summary This chapter describes the procedures to characterize the modification activity of type II and IIs MTases. The chapter also discusses purification of the MTase, the design of oligodeoxynucleotide substrates, the methylation of natural and synthetic substrates, fidelity of sequence recognition, and the identification of the modified base and the determination of its position within the recognition sequence. The best characterized MTases are those from type II and type IIs R-M systems. The ENases and MTases from these systems function as independent proteins encoded by separate genes, and they are widely used in the laboratory for recombinant DNA manipulation. The ENases require Mg 2+ and generally act only on unmodified DNA. The MTases require AdoMet and act on unmodified DNA or on DNA that is already modified on one strand. The enzymes recognize specific 4- to 8-nt sequences within dsDNA; for type II systems the sequences are symmetric, and for type IIs systems the sequences are usually asymmetric. Modification affects nucleotides within the recognition sequence and probably prevents endonucleolytic cleavage by steric hindrance.