In Vitro Transcription Translation and Analysis of Translated Protein

The TNTĀ® T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI) is convenient for PTT as both transcription and translation are performed simultaneously in a single tube using the PCR product as a template. There are several ways to detect the newly synthesized proteins. The most common method is incorporation of radiolabeled amino acids (e.g., 35S-methionine or 13C-leucine). For separation of the translation products, appropriate SDS-PAGE conditions must be chosen for simultaneous detection of both full-length and smaller products. After electrophoresis, gels are dried and protein bands are detected using either X-ray film or a phosphorImager screen. In general, a wildtype sample will have a strong band at the expected size

Fig. 2 ELISA-PTT. Panel A: Schematic representation of an ELISA-based Protein Truncation Test (ELISA-PTT). Unlike conventional PTT, the test uses a solid-phase ELISA format. B, Binding tag (either biotin which is incorporated randomly along the sequence using misaminoacylated tRNAs or epitope tag incorporated at N-terminal) acts to immobilize the cell-free synthesized fragments on the well surface. N- and C-terminal epitopes are detected by corresponding antibodies allowing for an estimate of the relative amount of truncated protein while simultaneously controlling for the level of translation. Fluo-roTags (FL) provide an independent confirmation of truncation and estimate of the fragment size by using fluorescence readout of gels. Panel B: Detection of various truncation mutations in the APC gene using ELISA-PTT. Panel C: Validation of ELISA-PTT result by gel-based fluorescence PTT. WT is wild-type, C1-C3 are mutant homozygous DNA samples from cell lines, and P1-P4 are the heterozygous DNA samples from patients prediagnosed with FAP. BL1 corresponds to a cell-free translation performed lacking both the added tRNAs and DNA. BL2 corresponds to a cell-free translation performed lacking only the added DNA. MW is molecular weight markers. The asterisk indicates the position of an autofluorescent protein band present in the cell-free translation extract. (View this art in color at www.dekker.com.)

of the full-length translation product compared to bands corresponding to smaller proteins. Ideally, these weaker bands correspond to truncated protein fragments derived from mutant template. However, weaker bands can also originate from internal weak translation initiation sites (AUG) and proteolytic degradation products. In some cases these bands can obscure the analysis and/or detection of the truncated fragments derived from a mutant template.

In addition to radiolabeling, methods have been reported which use nonisotopic detection. In one approach, biotin was incorporated during translation using biotin-lysine-tRNA and was detected by Western blotting.1-10-1 Similarly, Kahmann and coworkers1-11-1 used Western blotting to detect engineered epitope tags. Radioisotope labeling can also be eliminated by incorporating fluorescent labels using fluorescently labeled rn 12 13]

tRNAs.r , , J Although these nonisotope-based methods have clear advantages compared to radioactive detection, they still suffer from the intrinsic throughput problems associated with electrophoresis. In order to overcome these limitations, ELISA-PTT has been developed which provides higher throughput and lower cost because it circumvents electrophoresis.1-9-1

The basic ELISA-PTT approach is illustrated in Fig. 2A. Specially designed primers, which incorporate N- and C-terminal epitopes, are used for amplification of target sequences. In addition to these epitopes, additional tags can be incorporated randomly along the protein chain for detection and capture purposes. This is accomplished using mis-aminoacylated tRNAs (e.g., biotin-lysine-tRNA or/and BODIPY-lysine-tRNA) which are added to the reaction mixture. A capture tag can also be incorporated into the protein by using a specially designed primer. After translation, the test proteins are captured in a single-well of a microtiter plate, and the N- and C-terminal epitope tags are detected using appropriate antibodies. The signals obtained are used to compare the total amount of target protein captured (N-terminal signal) vs. the fraction that is full length (i.e., has a C-terminus).

The results of ELISA-PTT for the APC gene are shown in Fig. 2B. In this experiment, DNA derived from normal controls, familial adenomatous polyposis (FAP) patients as well as cell lines with known mutations in the APC gene was analyzed. The C/N terminal ratio of wild type was normalized to 100% and the values obtained for all other samples are expressed as a fraction of the wild type. For cell line DNA (homozygous APC mutant), the C/N terminal ratio was close to 0%, as expected. The C/N terminal ratio for heterozygote samples derived from individuals with FAP ranged from 37% to 47% relative to the wild type (Fig. 2B). It is possible to validate the ELISA-PTT results and localize the mutation within the protein using the fluorescent labels incorporated into

Table 1

Top ten diseases where PTT is applicable

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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