Reconstitution of proteins

Ruohola, H., Kabcenell, A.K., and Ferro-Novick, S. (1988). Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast the acceptor Golgi compartment is defective in the sec23 mutant. J Cell Biol 107 1465-1476. 

Protocols are presented below for the reconstitution of nuclear import of proteins and snRNPs using cell extracts as a source of transport factors and for the reconstitution of protein import using pure recombinant transport factors. 

The solubility of phospholipid membranes by non-ionic detergents is a basic technique in membrane biochemistry. Non-ionic rather than ionic surfactants are usually the first choice in biomembrane work when the preservation of structure and function of membrane proteins is desired. Two classes of non-ionic surfactants have found broad application, namely the n-alkylglycosides and the poly(oxyethylene)-n-alkyl- and acyl ethers. The former compounds are sometimes preferable for the reconstitution of proteins into closed membrane vesicles, due to their large critical micelle concentrations (cmc), while better protection against protein denaturation is usually achieved by the latter class of surfactants [1,2]. 

Studies on the effect of a number of antibiotics on chloramphenicol binding to ribosomes, and on reconstitution of protein synthetic activity by hybrid ribosomes from sensitive and resistant subunits, were carried out to confirm that the site of action of amicetin, sparsomycin, blasticidin S and streptogramin B is on the 50 S ribosome subunit 9a, Confirmation of the inhibitory effect of two antibiotics of the streptogramin family on... 

Product recoveiy from reversed micellar solutions can often be attained by simple back extrac tion, by contacting with an aqueous solution having salt concentration and pH that disfavors protein solu-bihzation, but this is not always a reliable method. Addition of cosolvents such as ethyl acetate or alcohols can lead to a disruption of the micelles and expulsion of the protein species, but this may also lead to protein denaturation. These additives must be removed by distillation, for example, to enable reconstitution of the micellar phase. Temperature increases can similarly lead to product release as a concentrated aqueous solution. Removal of the water from the reversed micelles by molecular sieves or sihca gel has also been found to cause a precipitation of the protein from the organic phase. 

Goloubinoff, P., Christeller, J.T., Gatenby, A.A., Lorimer, G.H. (1989). Reconstitution of active dimeric ribulosebiphosphate carboxylase from an unfolded state depends on two chaperonin proteins and magnesium ATP. Nature 342, 844-889. 

Thus three lines of evidence define the rapidly dissociating receptor as the LR complex. Conditions known to uncouple R from G--first, guanine nucleotide and second, pertussis toxin—produce LR third, reconstitution of G protein restores receptor affinity, sensitivity to guanine nucleotide, and effector activation. In this sense, the ligand and binding behavior of this system is analogous to that of the beta-adrenergic receptor, where the LR and LRG complexes have already been studied with purified proteins and reconstituted membrane preparations (2,i0). 

When the histone octamer is mixed with purified, double-stranded DNA, the same x-ray diffraction pattern is formed as that observed in freshly isolated chromatin. Electron microscopic studies confirm the existence of reconstituted nucleosomes. Furthermore, the reconsti-mtion of nucleosomes from DNA and histones H2A, H2B, H3, and H4 is independent of the organismal or cellular origin of the various components. The histone HI and the nonhistone proteins are not necessary for the reconstitution of the nucleosome core. 

The ion channel receptors are relatively simple in functional terms because the primary response to receptor activation is generated by the ion channel which is an integral part of the protein. Therefore, no accessory proteins are needed to observe the response to nicotinic AChR activation and the full functioning of the receptor can be observed by isolating and purifying the protein biochemically and reconstituting the protein in an artificial lipid membrane. In contrast, the G-protein-coupled receptors require both G-proteins and those elements such as phospholipase-C illustrated in Fig. 3.1, in order to observe the response to receptor activation (in this case a rise in intracellular calcium concentration resulting from the action of IP3 on intracellular calcium stores). 

Solubilization of an active H,K-ATPase is also a prerequisite for reconstitution of the enzyme into liposomes. With these H,K-ATPase proteoliposomes it is then possible to study the transport characteristics of pure H,K-ATPase, without the interference of residual protein contamination that is usually present in native vesicular H,K-ATPase preparations. Rabon et al. [118] first reported the reconstitution of choleate or n-octylglucoside solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. The enzyme was reconstituted asymmetrically into the proteoliposomes with 70% of the pump molecules having the cytoplasmic side extravesicular. In the presence of intravesicular K, the proteoliposomes exhibited an Mg-ATP-dependent H transport, as monitored by acridine orange fluorescence quenching. Moreover, as seen with native H,K-ATPase vesicles, reconstituted H,K-... 

Allen JR, SA Ensign (1997) Characterization of three protein components required for functional reconstitution of the epoxide carboxylase multienzyme complex from Xanthobacter strain Py2. J Bacterial 179 3110-3115. 

Ruan Z-S, V Anantharam, IT Crawford, SV Ambudkar, SY Rhee, MY Allison, PC Maloney (1992) Identification, purification, and reconstitution of OxlT, the oxalate formate antiport protein of Oxalobacter formigenes. J Biol Chem 267 10537-10543. 

Pereira MM, Carita JN, Teixeira M. 1999. Membrane-bound electron transfer chain of the ther-mohalophilic bacterium Rhodothermus marinus Characterization of the iron- sulfur centers from the dehydrogenases and investigation of the high-potential iron- sulfur protein function by in vitro reconstitution of the respiratory chain. Biochemistry 38 1276. 

The mechanisms of the oscillations in biomembranes have been explained based on the gating of membrane protein called an ion channel, and enormous efforts have been made to elucidate the gating process, mainly by reconstitution of channel proteins into bilayer membranes [9-11]. 

In the M. capsulatus (Bath) system, all three components are necessary to obtain turnover with NADH as the reductant (57). With the M. trichosporium OB3b system, protein B is apparently not required (27). Instead, in this latter system, protein B increases the initial rates of the catalytic hydroxylation reaction (27). Catalysis can be achieved by means of a shunt pathway with hydrogen peroxide and Hox alone from both organisms (58-60). The efficiency of the shunt pathway, however, varies significantly. With M. trichosporium OB3b, alcohol yields greater than those obtained with the completely reconstituted system have been observed (58). Furthermore, upon addition of protein... 

Protein fragment complementation assays are based on an enzyme reassembly strategy whereby a protein-protein interaction promotes the efficient refolding and complementation of enzyme fragments to restore an active enzyme. The approach was initially developed using the reconstitution of ubiquitin as a sensor for protein-protein interactions (Johnsson and Varshavsky, 1994). Ubiquitin is a 76 amino acid protein that... 

---