Protein reaction system

Figure 9.3 Schematic illustration of the electrophoretic transfer of proteins in the chromatophoresis process. After being eluted from the HPLC column, the proteins were reduced with /3-mercaptoethanol in the protein reaction system (PRS), and then deposited onto the polyacrylamide gradient gel. (PRC, protein reaction cocktail). Reprinted from Journal of Chromatography, 443, W. G. Button et al., Separation of proteins by reversed-phase Mgh-performance liquid cliromatography , pp 363-379, copyright 1988, with permission from Elsevier Science.

Fig. 1. Reconstruction of the cell-free protein synthesizing system with the partially purified wheat germ extracts. Control normal wheat germ cell-free system, (I) 0 - 40 % ammonium sulfate fraction 3 pi, 40 - 60 % ammonium sulfate fraction 4 pi, and ribosome 3 pi were added to 25 pi reaction mixture, (II) 0-40 % ammonium sulfate fraction 4 pi, 40 - 60 % ammonium sulfate fraction 4 pi, and ribosome 1.5 pi were added to 25 pi reaction mixture.

This equation illustrates the components of a competitive protein binding assay system. That is, the reaction system contains both radioactive and non-radioactive free ligand (P and P) and both radioactive and non-radioactive protein bound ligand (P Q and PQ). This type of assay assumes that binding protein will have the same affinity for the labeled or non-labeled material that is being measured. Although this assumption is not always completely valid, it usually causes no problems of consequence with most radioassays or radioimmunoassays. 

Schilling, M., F. Patett et al. (2007). Influence of solubility-enhancing fusion proteins and organic solvents on the in vitro biocatalytic performance of the carotenoid cleavage dioxygenase AtCCDl in a micellar reaction system. Appl. Microbiol. Biotechnol. 75(4) 829-836. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Furukawa, K., Mizushima, N., Noda, T., and Ohsumi, Y. A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes, J Biol Chem 2000, 275, 7462-7465. 

Antibody coverage of the human proteome is estimated to be about 5 to 10% of all human proteins and isoforms (Valle and Jendoubi, 2003). A major bottleneck in the use of protein expression arrays is the lack of such a comprehensive set of these capture agents (Hanash, 2003). Since an equivalent of the polymerase chain reaction (PCR) process for mass amplification of low abxmdant proteins does not exist, the remaining library of proteome capture ligands will need to be generated by other means such as recombinant protein expression systems (Cahill, 2001). 

The Transdirect insect cell is a newly developed in vitro translation system for mRNA templates, which utilizes an extract from cultured Spodoptera fru iperda 21 (S 21) insect cells. An expression vector, pTDl, which includes a 5 -imtranslated region (UTR) sequence from a baculovirus polyhedrin gene as a translational enhancer, was also developed to obtain maximum performance from the insect cell-free protein synthesis system. This combination of insect cell extract and expression vector results in protein productivity of about 50 pg per mL of the translation reaction mixture. This is the highest protein productivity yet noted among commercialized cell-free protein synthesis systems based on animal extracts. 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

The major drawback of this reaction system is the high energy and equipment costs due to the use of high pressures. In addition, the use of supercritical carbon dioxide can have adverse effects on enzymes, for example, by decreasing the pH of the microenvironment of the enzyme, by the formation of carbamates owing to covalent modification of free amino groups at the surface of the protein and by deactivation during pressurisation-depressurisation cycles [4]. 

There are many concepts one can transfer from heterogeneous catalyses to enzymatic catalyses and vice versa. In addition, the complexity of the previously reported system is of the same order as that encountered in simple enzymatic processes. Again the available indications point out that the knowledge of protein reactions can be advanced at a faster rate if one uses all the methods available not only from experiment but also from theory, including quantum mechanics. 

Biotin and biotin analogs Infant formula Protein precipitation using concentrated hydrochloric acid neutralization with 6 M NaOH lipid extraction with n-hexane Precolumn Microsorb C18 (15 X 4.6 mm, 5 jam Rainin). Analytical Microsorb C18 (250 X 4.6 mm, 5 /zm Rainin). Isocratic 100 mM phosphate buffer, pH 7.0 + methanol (80 + 20, v/v). 0.4 ml/min. Postcolumn reaction system UV absorbance at 220 nm followed by streptavidin-fluorescein isothiocyanate (2.0 mg/L) knitted open tubular reaction system (10.0 m x 0.5-mm ID) at a flow rate External standardization. 184 Linear range = 0.08-1.00 fjM biotin. LoD = 0.02 /zM or 97 pg biotin at SNR = 3. Repeatability CV 3.5% for biotin in infant formula. 

Loss of Available Lysine in Protein in a Model Maillard Reaction System... 

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