Expression and purification of full length mouse metal response element binding transcription factor-1 using Pichia pastoris

K-Ras4B, a small GTPase and a key oncogene, plays a central role in the early steps of signal transduction from activated receptor tyrosine kinases by recruiting its downstream effectors to the cell membrane. Specific posttranslational modifications of K-Ras4B, including the addition of C-terminal farnesyl and methyl groups, mediate its proper membrane localization and signaling activity. The mechanism and molecular determinants underlying this selective membrane localization and molecular interactions with its many regulators and downstream effectors are largely unknown. Preparative amounts of the posttranslationally processed K-Ras4B protein are necessary to carry out structural, functional, and cell biological studies of this important oncogene. In this work we describe a simple and efficient method for synthesis of milligram quantities of functionally active, fully processed K-Ras4B. Using this preparation, we observe K-Ras4B dimerization in vitro; this has not been observed previously and could be important for its activity, membrane anchoring, and translocation between different cellular membranes.     

Role of the zinc fingers of HIV-1 nucleocapsid protein in maturation of genomic RNA

The nucleocapsid protein of HIV-1 consists of two basic amino acid regions and two zinc fingers. We investigated the requirement of these domains for the structural conversion of a 39mer RNA covering the dimerization initiation site by using three peptides; wild-type NCp7, a mutant in which the two zinc fingers are mutated, and another mutant in which the two zinc fingers are deleted. The two mutants exhibited similar conversion activities to each other, which were lower than that of the wild-type, indicating that the two basic regions exhibit some activity for RNA chaperone, as we suggested before, and the zinc fingers enhance the efficiency of this activity.