Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

High levels of protein expression using different mammalian CMV promoters in several cell lines.

With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Triclosan/pyrophosphate dentifrice: dental plaque and gingivitis effects in a 6-month randomized controlled clinical study.

A double-blind, parallel, randomized and controlled clinical trial was conducted on 186 subjects over six months to assess the effects of a 0.28% triclosan/5% pyrophosphate (with NaF/silica) dentifrice on dental plaque and gingivitis as compared to a NaF/silica negative control dentifrice. An initial examination was performed to assess the health of the oral soft and hard tissues and to measure plaque (by Turesky modified Quigley-Hein Plaque Index), gingivitis (by Löe-Silness Gingival and Ainamo and Bay Gingival Bleeding [GBI] indices). Only those subjects with a GBI score > or = 5 were accepted into the study. Each enrolled subject received an oral prophylaxis and was requested to brush and floss twice per day with the negative control NaF/silica dentifrice. After one month, the subjects were recalled and a baseline examination was performed for each of the previously described parameters. Following the baseline examination, the subjects received another oral prophylaxis. The subjects were then separated by gender and by baseline GBI scores of < or = 7 or > 7 and arrayed by the changes in GBI bleeding sites from initial to baseline. Within strata, subjects were randomly assigned to brush twice per day with either the triclosan/pyrophosphate dentifrice or the negative control dentifrice. The subjects were subsequently examined for all of the above-described parameters following use of the test dentifrices for five weeks, three and six months. The data generated in this trial were analyzed using an analysis of covariance on all indices for all subjects completing the examinations. The results from this study demonstrated that the use of the triclosan/pyrophosphate dentifrice resulted in statistically significant reductions of dental plaque compared to the control by 10% (p < 0.05), 15.4% (p < 0.01) and 13.9% (p < 0.01) at five weeks, three and six months, respectively. However, there were no statistically significant differences between the test dentifrices for any of the gingivitis or gingival bleeding evaluations throughout the study. Based on 1) the fact that subjects possessed plaque-induced gingivitis in this clinical study, 2) the similarity in the magnitude of the plaque reductions observed from the triclosan/pyrophosphate dentifrice relative to those reported for other triclosan-containing dentifrices, 3) the similarity in the dose of triclosan relative to other triclosan dentifrices, and 4) the reported magnitude of gingivitis reductions from other triclosan-containing dentifrices, these findings were unexpected. Possible explanations of these results are that the triclosan/pyrophosphate dentifrice may be uniquely different from other triclosan dentifrices relative to its effects on gingivitis, or alternatively, the clinical design utilized here may not be optimized for triclosan/pyrophosphate dentifrice.