Strategy for overexpression and structural analysis of GumB, an outer membrane lipoprotein

JACOBS M, CARO SOLÍS C, BIANCO MI, SALINAS SR AND IELPI L

Puerto Madryn, Chubut

Congreso; Reunión Anual – Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2010

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

Membrane proteins play essential roles in different cellular processes as signaling among cells, transport across membranes, and energy transduction. Despite their importance, there are few known 3D structures and less is known about their mechanism of action compared to soluble proteins. The lack of knowledge in this field is due to difficulties in obtaining enough amount of protein to carry out structural studies. Here we describe a strategy to resolve the structure of GumB, an outer membrane lipoprotein involved in secretion of xanthan, which is hard to overexpress. In initial overexpression assays, we found that GumB resulted toxic to the cells. After several trials, we found a condition with an appropriate level of expression. However, the amount of protein was not enough to set up crystallization screenings. At the moment, we are performing different purification protocols in order to obtain homogeneous pure GumB, in adequate amount suitable for the beginning of crystallization trials. As an alternative, we overexpressed, purified, and crystallized a form of GumB that lacks the signal peptide. Thus, it is overexpressed as a soluble protein in the cytoplasm (GumBc). Currently, we are working on the resolution of the 3 Å GumBc structure in order to use it as template for molecular replacement and to determine the structure of the lipidated form of GumB localized in the membrane.