Production of Recombinant Rhomboid Proteases.

Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.

Activity Assays for Rhomboid Proteases.

Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and Vmax to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein.

High-yield expression and functional analysis of Escherichia coli glycerol-3-phosphate transporter.

The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli mediates G3P and inorganic phosphate exchange across the bacterial inner membrane. It possesses 12 transmembrane alpha-helices and is a member of the Major Facilitator Superfamily. Here we report overexpression, purification, and characterization of GlpT. Extensive optimization applied to the DNA construct and cell culture has led to a protocol yielding approximately 1.8 mg of the transporter protein per liter of E. coli culture. After purification, this protein binds substrates in detergent solution, as measured by tryptophan fluorescence quenching, and its dissociation constants for G3P, glycerol-2-phosphate, and inorganic phosphate at neutral pH are 3.64, 0.34, and 9.18 microM, respectively. It also shows transport activity upon reconstitution into proteoliposomes. The phosphate efflux rate of the transporter in the presence of G3P is measured to be 29 micromol min(-1) mg(-1) at pH 7.0 and 37 degrees C, corresponding to 24 mol of phosphate s(-1) (mol of protein)(-1). In addition, the glycerol-3-phosphate transporter is monomeric and stable over a wide pH range and in the presence of a variety of detergents. This preparation of GlpT provides ideal material for biochemical, biophysical, and structural studies of the glycerol-3-phosphate transporter.

Secretion and processing of apolipoprotein A-I in the avian sciatic nerve during development.

Apolipoprotein A-I (apo A-I), a major apolipoprotein synthesized by liver and intestine to facilitate transport of plasma lipids as lipoproteins, has been detected also in the avian sciatic nerve. The mRNA and protein levels of apo A-I have been shown to increase during the period of rapid myelination (LeBlanc et al.: J Cell Biol 109:1245-1256, 1989). In order to assess the synthesis of apo A-I protein and the processing of apo A-I isoforms during development, endoneurial slices of avian sciatic nerves from chicks during active myelination at 15 and 17 days embryonic and 1 day posthatch age were incubated with [35]S-methionine. The incubations were fractionated into secreted and intracellular fractions, and incorporation of the label was assessed for apo A-I protein. The pattern of labeling of Po protein, as a marker of myelination, was also determined in the intracellular and compact myelin fractions. Methionine incorporation into Po protein was highest in the intracellular compartment at the 15-day embryonic stage and decreased thereafter, with a corresponding increase in the myelin fraction. During these developmental periods, the levels of nascent apo A-I increased in both the secreted and intracellular fractions. The synthesis of apo A-I specifically increases in the secreted fraction compared with total protein synthesis. The processing of the pro-apo A-I is also developmentally regulated. In the intracellular compartment, there are approximately equal proportions of the acidic and basic isoforms. However, with increasing age, a higher proportion of the apo A-I is secreted as acidic isoforms. It is concluded that the secretion and processing of apo A-I is developmentally regulated in the chick sciatic nerve, in parallel with the process of active myelination.

Biosynthesis and compartmentalization of Po, apolipoprotein A-I, and lipids in the myelinating chick sciatic nerve.

Myelin deposition in developing chick sciatic nerve is associated with rapid synthesis of lipids, the major myelin protein Po and apo A-I, a major constituent of plasma lipoproteins. In order to understand possible roles of apo A-I in myelin assembly the synthesis and appearance of Po, apo A-I and lipids was studied in an intracellular fraction, an intralamellar fraction thought to be related to, or derived from, myelin and compact myelin from rapidly myelinating sciatic nerve of 1 day chicks. Incorporation with methionine or pulse-chase experiments indicated that initial synthesis of Po occurs in the intracellular fraction followed by movement to the intralamellar fraction and myelin. Incorporation of labelled oleate into phospholipids suggested that initial synthesis occurs in the intracellular and intralamellar fractions with slow movement to myelin. Incorporation of labelled galactose into cerebrosides suggested that initial synthesis occurs partially in myelin with slow loss from this fraction to the intralamellar fraction. However, incorporation of methionine into apo A-I indicated that initial synthesis occurred in the intracellular fraction with some transfer to the intralamellar fraction and secretion of a major portion into the incubation medium. It is concluded that the subcellular distribution of nascent apo A-I is not well coordinated with the distribution of other nascent constituents of the myelin membrane. The accumulation of nascent Po, phospholipids and cerebrosides in the intralamellar fraction compared to compact myelin suggests that this fraction may play a role as a precursor membrane or as a storage site for assembly of myelin constituents into compact myelin.