Molecular Cloning and Expression of Starfish Protein Kinase C Isoforms

Molecular Cloning, Expression, and Characterization of Starfish DNA (Cytosine-5)-methyltransferases

“…His 10 -tagged sDnmt1ct polypeptides were purified from E. coli BL21 (DE3) harboring this expression plasmid by Ni-NTA agarose (Qiagen, Hilden, Germany) column chromatography under denaturing conditions, as described previously for the starfish PKC C-terminal domain. 29) sDnmt1ct was further purified by SDS-PAGE, and the band corresponding to sDnmt1ct was cut out to recover the polypeptide by electro-elution. Antiserum against sDnmt1ct was raised by immunizing a rabbit, as described previously for antiserum against the C-terminal domain of starfish cPKC.…”

“…His 6 -tagged sDnmt1 was purified to homogeneity from overexpressing insect cells by metalchelate affinity chromatography, essentially as described previously for starfish aPKC. 29) Cells were suspended in 100 mL of the base buffer (20 mM potassium phosphate buffer pH 7.4/300 mM KCl/0.5% Brij35) and disrupted by sonication. After centrifugation at 20;600 Â g for 20 min, the supernatant was applied to a Ni-NTA agarose column (bed volume, 100 mL) that was first equilibrated with base buffer containing 10 mM imidazole and 10 mM DTT.…”

Mechanisms for Insulin Resistance: Common Threads and Missing Links

“…The PKC kinases belong to a larger family of AGC kinase that include a broad array of serine/threonine kinases and are themselves further subdivided into three categories: conventional PKC’s (cPKC: α, βI, βII and γ) that require both calcium and diacylglycerol for activation, novel PKC’s (nPKC: δ, ε, η, and θ) that require only diacylglycerol and atypical PKC’s (aPKC: ζ and λ) that require neither calcium nor diacylglycerol. Of interest, sequence analyses suggest that the atypical PKC’s may in fact be the most ancient, suggesting that as biological systems became more complex, PKC’s evolved into additional signaling roles (Miyake et al, 2009). …”

Cloning, Heterologous Expression, and Enzymatic Characterization of Cathepsin L from Starfish (Asterina pectinifera)

“…19,20) They were used within 1 week of collection, without food. Total RNA was extracted from various tissues (muscle, gonad, tubefeet, liver, stomach, and body-rind) of three animals (average width, 10.65-11.05 cm) with oligo (dT) 18 and random hexamer primers using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, IN), in following the manufacturer's instructions.…”