T7 Tag Peptide

An autoantibody produced by a hybridoma derived from a viable motheaten mouse was isolated and found to have moderately high binding affinity for nucleic acids and specific preference for quadruplex DNAs. Polymerase chain reaction primers were designed to link the cloned parental antibody variable region fragments together in a subcloning vector. This single-chain variable fragment construct was then subcloned into the T7 promoter-driven expression vector pET22b(+). The construct contains (N- to C-terminal) a pelB leader sequence, variable heavy chain, glycine-serine polylinker, variable light chain, and biotin mimic peptide "strep-tag" sequence (pelB-VH-linker-VL-strep-tag). The ca. 29 kDa protein was expressed, exported to the periplasmic space of NovaBlue (DE) Escherichia coli, and purified by streptavidin affinity chromatography by binding the fused strep-tag peptide. The specificity of the purified single-chain variable fragment (scFv) for quadruplex and duplex DNAs was evaluated by a radioimmunofilterbinding assay. It retained about 10-fold higher affinity for quadruplexes relative to duplex DNA, a reduction of ca. 4-fold from the relative preferences of the parent IgG. The complementary-determining regions contain sequences that are homologous to or conservatively divergent from the key DNA-binding helix-turn-helix-forming motifs of Myb/RAP1 family telomeric DNA-binding proteins (1-3). The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.

A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (P(mxaF)), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S. tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37 degrees C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C(8)), with K(m) and k(cat) values of 14 +/- 1.08 microM and 1,245 +/- 42.3 S(-1), respectively.

Here we report on the overexpression and in vitro characterization of a recombinant form of ExoM, a putative beta1-4 glucosyltransferase involved in the assembly of the octasaccharide repeating subunit of succinoglycan from Sinorhizobium meliloti. The open reading frame exoM was isolated by PCR and subcloned into the expression vector pET29b, allowing inducible expression under the control of the T7 promoter. Escherichia coli BL21(DE3)/pLysS containing exoM expressed a novel 38-kDa protein corresponding to ExoM in N-terminal fusion with the S-tag peptide. Cell fractionation studies showed that the protein is expressed in E. coli as a membrane-bound protein in agreement with the presence of a predicted C-terminal transmembrane region. E. coli membrane preparations containing ExoM were shown to be capable of transferring glucose from UDP-glucose to glycolipid extracts from an S. meliloti mutant strain which accumulates the ExoM substrate (Glcbeta1-4Glcbeta1-3Gal-pyrophosphate-polyprenol). Thin-layer chromatography of the glycosidic portion of the ExoM product showed that the oligosaccharide formed comigrates with an authentic standard. The oligosaccharide produced by the recombinant ExoM, but not the starting substrate, was sensitive to cleavage with a specific cellobiohydrolase, consistent with the formation of a beta1-4 glucosidic linkage. No evidence for the transfer of multiple glucose residues to the glycolipid substrate was observed. It was also found that ExoM does not transfer glucose to an acceptor substrate that has been hydrolyzed from the polyprenol anchor. Furthermore, neither glucose, cellobiose, nor the trisaccharide Glcbeta1-4Glcbeta1-3Glc inhibited the transferase activity, suggesting that some feature of the lipid anchor is necessary for activity.