Protein Expression and PuriWcation 47 (2006) 303–310 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2005.11.005 Gi/o proteins: Expression for direct activation enquiry Lorenzo Di Cesare Mannelli a,¤,1 , Alessandra Pacini b,1 , Annarita Toscano b , Martina Fortini c , Debora Berti c , Carla Ghelardini a , Nicoletta Galeotti a , Piero Baglioni c , Alessandro Bartolini a a Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50134 Florence, Italy b Department of Anatomy, Histology and Forensic Medicine, Anatomy Section, University of Florence, Viale Morgagni 85, 50134 Florence, Italy c Department of Chemistry and CSGI, University of Florence, Via della Lastruccia 3, 50019 Sesto F.no, Italy Received 8 September 2005, and in revised form 7 November 2005 Available online 5 December 2005 Abstract G protein-mediated pathways are fundamental mechanisms of cell signaling. In this paper, the expression and the characterization of the  i1 ,  i3 ,  o1 ,  1 , and  2 subunits of the human G protein are described. This approach was developed to evaluate the G protein activa- tion proWle of new compounds. pCR-TOPO T7 vectors, engineered to contain the target sequences, were used to transform Escherichia coli competent cells. Subunits were over-expressed in a preparative scale as fusion proteins with a six-histidine tag, and subsequently puri- Wed by metal chelate chromatography. Afterward, the His-tag was removed by enterokinase digestion, and the secondary structures of the recombinant subunits were analyzed by circular dichroism. To assess the functionality of the subunits, the rate of GTP hydrolysis and GTPS binding were evaluated both in the absence and in the presence of two modulators: the peptidic activator Mastoparan and the non-peptidic activator N-dodecyl-lysinamide (ML250). Tests were conducted on isolated -subunit and on heterotrimeric  complex, alone or reconstituted in phospholipidic vesicles. Our results show that recombinant subunits are stable, properly folded and, fully active, which makes them suitable candidates for functional studies.  2005 Elsevier Inc. All rights reserved. Keywords: Human Gi proteins; Escherichia coli expression; PuriWcation; Circular dichroism; Mastoparan; Receptor-independent G protein modulation; Direct G protein activator G proteins are an evolutionarily highly conserved group of molecules known as heterotrimeric guanine nucleotide- binding proteins. Numerous molecules, such as hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli, exert their eVect on cells by binding to hep- tahelical membrane receptors coupled to heterotrimeric G proteins. When a ligand interacts with a heptahelical recep- tor on the surface of the cell, the ligand either stabilizes or induces a conformation in the receptor that activates a het- erotrimeric G protein, composed of -, -, and -subunits, on the inner membrane surface of the cell [1]. According to the current knowledge, 17 genes encode for 23 mammalian G-subunits [2–4], 5 genes encode for G- subunits [5], and 12 genes encode for G-subunits [2,6]. All -subunits share biochemical and structural properties and each of them can be assigned to structurally and function- ally related groups [7,8]. Classically, G proteins are divided into four families based on similarity of their -subunits: G i/o , G s , G q/11 , and G 12/13 . When bound to GTP, G-subunits can regulate intracellular eVectors, such as adenylyl cyclase, phospholipase C  , K + , and Ca 2+ channels, and cyclic GMP phosphodiesterases. G protein - and -subunits are tightly associated, and form stable dimers that cannot be separated unless the pro- teins are denatured. Hence, from a functional point of view, they are considered as a single entity, the -dimer. The number of -dimers and, most signiWcantly, of oligomeric * Corresponding author. Fax +39 0554271280. E-mail address: lorenzo.mannelli@uniW.it (L. Di Cesare Mannelli). 1 These authors contributed equally to this work.