Affinity tagging

E. coli strain BL21(DE3) (F ompT ksdSafra ms ) gal dcm) (Novagen, Madison, WI, USA) was used as a host for recombinant OPH expression. Recombinant plasmids pTOH and pEOH that contain OPH gene fused with hexa-histidine affinity tag under trc and T7 promoter, respectively, as control vectors and pTTOH and pETOH that contain OPH gene fused with Tat signal sequence and hexa-histidine affinity tag under trc and T7 promoter, respectively, were used (Fig. 1). 

Fig.l. Gene maps of recombinant plasmids pTOH, pTTOH, pEOH, and pETOH. Abbreviations Pnc, trc promoter Px7, T7 and lac hybrid promoter Tat, twin-arginme TorA signal sequence of TMAO reductase OPH, organophosphoms hydrolase gene Hisg, hexa-histidine affinity tag. 

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

In vitro labeling of proteins using isotope-coded affinity tags... 

Figure 3.3. Structure of the ICAT reagent. The reagent contains a biotin affinity tag that is used to isolate ICAT-labeled peptides. The reagent also contains a linker that exists in a heavy (where X= deuterium) or light form (X= hydrogen) and a reactive group with specificity towards the thiol groups of cysteine residues. Figure adapted from Gygi et al. (1999).

Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999). Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994-999. 

Von Haller, P.D., Yi, E., Donohoe, S., Vaughn, K., Keller, A., Nesvizhskii, A.I., Eng, J., Li, X.J., Goodlett, D.R., Aebersold, R., Watts, J.D. (2003). The Application of New Software Tools to Quantitative Protein Profiling Via Isotope-coded Affinity Tag (ICAT) and Tandem Mass Spectrometry II. Evaluation of Tandem Mass Spectrometry Methodologies for Large-Scale Protein Analysis, and the Application of Statistical Tools for Data Analysis and Interpretation. Mol. Cell. Proteomics 2, 428 -42. 

Nickel affinity chromatography was chosen as the primary purification technique because it is a fast and reliable one-step assay and purified complexes can often be used in downstream applications without the necessity of removing the polyhistidine tag. In addition, the polyhistidine tag is smaller than many other affinity tags targeted by commercially available affinity resins and, in most cases, does not seem to interfere with the structure and function of the recombinant protein. 

To provide the reader with a more complete picture, this section describes the use of affinity tags other than a Hisg-tag which were also used successfully for purification of eIF3 and its binding partners (Asano et ah, 2000 Valasek et ah, 2003). 

Figure 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at particular functional groups and the biotin affinity tag is used to isolate labeled molecules using immobilized (strept)avidin.

Figure 28.10 Trifunctional label transfer agents contain two arms with terminal reactive groups and a third arm with a label or affinity tag, such as a biotin group. One of the reactive groups typically is thermoreactive to couple with bait proteins, while the second reactive group usually is photoreactive. The thermoreactive arm has a cleavable cross-bridge to facilitate release of the captured protein and transfer of the label of affinity tag to it.

Figure 28.12 Sulfo-SBED first is used to label a bait protein through reaction of the sulfo-NHS ester with available amine groups on the protein, yielding an amide bond linkage. This labeled bait protein then is added to a sample containing proteins that potentially could interact with the bait. After an incubation period, the sample is exposed to UV light to photoactivate the phenyl azide group. This reaction causes any interacting prey proteins to be crosslinked with the bait protein, forming a complex containing a biotin affinity tag.

Figure 28.14 A trifunctional label transfer reagent containing a thiol-reactive pyridyl disulfide group, a photo-reactive phenyl azide, and a biotin affinity tag. This compound can be used to label bait proteins through available thiol groups and capture interacting prey proteins by photoreactive conjugation.

Figure 28.15 Two similar label transfer reagents containing a thiol-reactive methanethiolsulfonate group to label bait protein through available sulfhydryls, a tetrafluorophenyl azide group for high-efficiency photoreac-tive conjugation with interacting prey proteins, and a long biotin affinity tag.

Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H., and Xu, M.-Q. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192, 271-281. 

Fauq, A.H., Kache, R., Khan, M.A., and Vega, I.E. (2006) Synthesis of acid-cleavable light isotope-coded affinity tags (ICAT-L) for potential use in proteomic expression profiling analysis. Bioconjugate Cbem. 17, 248-254. 

Han, B.N., Stevens, J.F., and Maier, C.S. (2007) Design, synthesis, and application of a hydrazide-func-tionalized isotope-coded affinity tag for the quantification of oxylipid-protein conjugates. Anal. Chem. 79(9), 3342-3354. 

Hansen, K.C., Schmitt-Ulms, G., Chalkley, R.J., Hirsch, J., Baldwin, M.A., and Burlingame, A.L. (2003) Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography. Mol. Cell. Proteomics 2, 299-314. 

Lee, S., Young, N.L., Whetstone, P.A., Cheal, S.M., Benner, W.H., Lebrilla, C.B., and Meares, C.F. (2006) A method to site-specifically identify and quantitate carbonyl end products of protein oxidation using oxidation-dependent element coded affinity tags (O-ECAT) and nanoLiquid chromatography Fourier transform mass spectrometry./. Proteome Res. 5(3), 539-547. 

Li, J., Steen, H., and Gygi, S.P. (2003) Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents. The yeast salinity stress response. Mol. Cell. Proteomics 2, 1198-1204. 

Lu, Y., Bottari, P., Turecek, F., Aebersold, R., and Gelb, M.H. (2004) Absolute quantification of specific proteins in complex mixtures using visible isotope-coded affinity tags. Anal. Chem. 76, 4104 flll. 

Meares, C.F., Whetstone, P.A., Corneillie, T.M., Butlin, N.G. (2007) Element-coded affinity tags. US... 

Turecek, F. (2002) Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis. J. Mass Spectrom. 37, 1-14. 

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