[43] Purification, properties, and analysis of yARL3 (original) (raw)

Methods in Enzymology, 2001

Abstract

ABSTRACT This chapter describes a method to prepare recombinant y adenosine diphospahte (ADP) ribosylation factors (ARF)-like proteins (ARL)3 from Escherichia coli using a pET-based vector. Recombinant yARL3 protein is isolated in a functional form from the soluble fraction of the bacterial lysate using a rapid two-step procedure involving Ni2+-agarose chromatography followed by gel filtration. This chapter also describes assays to quantify guanine nucleotide binding and guanosine tri phosphate (GTP) hydrolysis. The ability of purified recombinant yARL3 to bind GTPTS is determined by a filter trapping method. GTP hydrolysis is determined by binding [α-32p]GTP to recombinant yARL3 protein.

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