Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques

Transcription and polyadenylation processes during early development of quail embryo

ABSTRACT Transcription of tRNA and mRNA occurs in quail as early as during cleavage while rRNA transcription becomes measurable during blastulation. The polyadenylated fraction content in newly synthesized RNA amounting to 6 % during cleavage and blastulation decreases to 3 % during gastrulation. Up to 3/4 adenylic residues incorporated into RNA during cleavage are accumulated in the polyadenylated molecules mainly in the form of RNAse-resistant tracts.</jats:p