Expression and purification of untagged Glnk proteins from actinobacteria

Abstract

The PII protein family constitutes one of the most conserved and well distributed family of signal transduction proteins in nature. These proteins play key roles in nitrogen and carbon metabolism. PII function has been welldocumented in Gram-negative bacteria. However, there are very few reports describing the in vitro properties and function of PII derived from Gram-positive bacteria. Here we present the heterologous expression and efficientpurification protocols for untagged PII from three Actinobacteria of medical and biotechnological interest namely: Mycobacterium tuberculosis, Rhodococcus jostii and Streptomyces coelicolor. Circular dichroism and gel filtrationanalysis supported that the purified proteins are correctly folded. The purification protocol described here will facilitate biochemical studies and help to uncover the biochemical functions of PII proteins in Actinobacteria.