In vitro compartmentalization

Griffiths, A.D. and Tawfik, D.S. (2003) Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. The EMBO Journal, 22, 24-35. 

Agresti, J.J., Kelly, B.T., Jaschke, A., and Griffiths, A.D. (2005) Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization. Proc. Natl. Acad. Sci. USA 102,16170-16175. 

A.D. Griffiths and D.S. Tawfik Directed Evolution of an Extremely Fast Phospho-triesterase by In Vitro Compartmentalization. Embo J. 22, 24 (2003). 

K. Bemath, M. Hai, E. Mastrobattista, A.D. Griffiths, S. Magdassi, and D.S. Tawfik In Vitro Compartmentalization by Double Emulsions Sorting and Gene Enrichment by Fluorescence Activated CeU Sorting. Anal. Biochem. 325, 151 (2004). 

Figure 8.3. Probability of finding at least one antibody than recognizes any given epitope as a function of repertoire size and threshold Kd value.5 [Reprinted, with permission, from A. D. Griffiths and D. S. Tawfik, Current Opinion in Biotechnology 2, 2000, 338-353. Man-Made Enzymes—From Design to In Vitro Compartmentalization . 2000 Elsevier Science Ltd.]...

Cellular compartmentalization has inspired a range of new, and potentially useful, in vitro systems that treat droplets of water as microreactors. These have been recently reviewed by Griffiths and coworkers.298 The first DNA-display experiments, using this idea, were reported by Tawfik and Griffiths, who developed an in vitro compartmentalization (IVC) method using water-in-oil emulsions, which can be cheaply manufactured from oil, emulsifier, and detergent. By carefully selecting the conditions, a DNA library can be spatially arrayed by... 

Figure 5. In vitro compartmentalization utilizes the aqueous core of water-in oil (w/o) or water/oil-in water (w/o/w) emulsions for in vitro translation. Proteins are expresses on the basis ofDNA in each emulsion and the catalytic activity ofprotein is evaluated. Here, the fluorescence intensity of each emulsion is correlated with the enzymatic activity. Therefore, the emulsions with high fluorescence intensity are sorted by FACS, thus obtaining a pool of DNAs encoding enzymes with high activity. 

Griffiths, A.D. Tawfik, D.S. Man-made enzymes—From design to in vitro compartmentalization. Curr. Opin. Biotechnol. 2000. II. 338-353. 

Gray GL, Power SD, Poulouse AJ (1995) Lipase from Pseudomonas Mendocina having cutinase activity. US Patent 5,389,536 Griffiths AD, Tawfik DS (2003) Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J 22 24-35 Gusakov AV, Sinitsyn AP, Berlin AG, Markov AV, Ankudimova NV (2000) Surface hydrophobic amino acid residues in cellulase molecules as a structural factor responsible for their high denim-washing performance. Enzyme Microb Technol 27 664-671... 

Extending the emulsion to a water-in-oil-in-water mixture allowed further refinement of the IVC concept. Compartmentalization of E. coli containing semm paraoxonase variants allowed the accumulation of fluorescent product to a point where it could be detected by FACS [57]. This approach was also used with in vitro transcription and translation to evolve /3-galactosidase activity from the Ebg gene [58]. 

Refinement and expansion of these steady-state mass balance approaches has led to the development of dynamic models which allow for estimation of the fraction absorbed as a function of time and can therefore be used to predict the rate of dmg absorption [37], These compartmental absorption and transit models (CAT) models have subsequently been used to predict pharmacokinetic profiles of drugs on the basis of in vitro dissolution and permeability characteristics and drug transit times in the intestine [38],... 

Johnson BM, Charman WN, Porter CJH (2003) Application of compartmental modelling to an examination of in vitro intestinal permeability data Assessing the impact of tissue uptake, P-glycoprotein, and CYP3A. Drug Metab Dispos 31 1151-1160. 

GastroPlus [137] and IDEA [138] are absorption-simulation models based on in vitro input data like solubility, Caco-2 permeability and others. They are based on advanced compartmental absorption and transit (ACAT) models in which physicochemical concepts are incorporated. Both approaches were recently compared and are shown to be suitable to predict the rate and extent of human absorption [139]. 

Figure 7.7. Schematic representation of gene selection by compartmentalization. Step 1 An in vitro transcription/translation reaction mixture containing a library of genes linked to a substrate for the reaction being selected is dispersed to form a water-in-oil emulsion with typically one gene per aqueous compartment. Step 2 The genes are transcripted and translated within their compartments. Step 3 Proteins (or RNAs) with enzymatic activities convert the substrate into a product that remains linked to the gene. Compartmentalization prevents the modification of genes in other compartments. Step 4 The emulsion is broken all reactions are stopped and the aqueous compartments are combined. Genes linked to the product are selectively enriched, then amplified, and either characterized (step 5) or linked to the substrate and compartmentalized for further rounds of selection (step 6). (Adapted from [39].)...

Schlesinger et al. 20) concluded, on the basis of in vitro rates of dimerization, that the dimerization of enzyme subunits in vivo would not be as rapid as observed unless the subunits were compartmentalized in the cell. The in vitro rate of dimerization seemed to be based upon reoxidation and dimerization of reduced monomers and showed a maximum at 65 /ig/ml with respect to protein concentration. The in vivo process may be rather different, however, and later studies by Schlesinger and Barrett 21) with unreduced monomers would seem to change this conclusion because their rates did not have a maximum with respect to protein concentration. 

---