1.Inhibition of plant cell proteolytic activities that degrade tubulin.
Morejohn LC, Bureau TE, Fosket DE. Cell Biol Int Rep. 1985 Sep;9(9):849-57.
The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose (Rosa sp. cv. Paul's scarlet) was examined by NadodecylSO4-polyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of alpha-subunit to beta-subunit, and low molecular weight polypeptides, one (approximately 32 Kd) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C- and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual alpha- and beta-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity.
2.Cannabinoid receptor 2 expression modulates Gβ1γ2 protein interaction with the activator of G protein signalling 2/dynein light chain protein Tctex-1.
Nagler M1, Palkowitsch L2, Rading S1, Moepps B3, Karsak M4. Biochem Pharmacol. 2016 Jan 1;99:60-72. doi: 10.1016/j.bcp.2015.09.017. Epub 2015 Sep 26.
The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gβγ, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G protein-coupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gβγ. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1-Gβγ-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gβγ interacts with Tctex-1, but competes with Tctex-1 in binding to Gβγ.
3.Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures.
Ganapathi TR1, Sunil Kumar GB, Srinivas L, Revathi CJ, Bapat VA. Plant Cell Rep. 2007 Sep;26(9):1575-84. Epub 2007 May 30.
Soybean cell suspension cultures were transformed using Agrobacterium tumefaciens harboring pHBS/pHER constructs to express hepatitis B surface antigen (HBsAg). The transformed colonies were selected and analyzed for the expression of HBsAg by PCR, reverse transcription (RT) PCR, Western blot and ELISA analysis. The maximum expression of 700 ng/g F.W. was noted in pHER transformed cells. The highest expressing colonies were used to initiate the cell suspension cultures and the expression of HBsAg was estimated periodically. The expression levels were reduced drastically in cell suspension cultures compared to the colonies maintained on semi-solid medium. Various parameters were studied to maximize the cell growth and to retain the expression levels. The supplementation of culture medium with a protease inhibitor, leupeptin hemisulfate could restore up to 50% of HBsAg expression in cell suspension cultures. This is the first report to investigate the possible cause and solution to the loss of recombinant protein expression levels in plant cell suspension cultures.
