Functional Characterization of Transgenic Lines

Prior to purification, a preliminary functional and molecular characterization of the TAPa-tagged proteins should be performed. For this, independent Arabidopsis trans-genic lines for each TAPa fusion should be obtained and fusion protein levels for each independent line should be compared using immunoblots against the myc epi-tope. Fusion expression levels may vary greatly among independent transgenic lines for the same TAPa fusion possibly reflecting positional effects at transcriptional level due to the transgene location [7]. Although one major band corresponding to the

TAPa-tagged protein should be expected, previous results have shown that other specific minor bands might appear, probably corresponding to truncated versions of the corresponding TAPa fusion. Indeed, additional bands use to correlate with a high level of transgene expression, suggesting that the higher the expression is, the more likely it is that the TAPa fusions are processed and truncated by nonspecific proteases. In addition, for some specific tagged proteins, fusion size might look much bigger than expected, according to its electrophoretic mobility. This anormal electrophoretic behavior might be due to a wide variety of PTMs, such as glycosyla-tion, ubiquitination, and sumoylation, among others [11]. On the other hand, slower mobility in denaturing gels has been shown to be inherent to specific target proteins [12, 13]. Isolation and determination of each band composition, upon TAPa procedure, should help to discriminate between these two possibilities.

Functional characterization of transgenic lines can be accomplished by performing a mutant phenotype complementation analysis. In the case where the TAPa transgenes were not transformed into the corresponding mutant background, study of potential gain-of-function phenotypes due to overexpression of the targeted proteins can provide insights into their functionality. In both cases, transgenic plants should be grown in the specific conditions reported for each genetic background and different parameters should be analyzed and compared to those in mutant and/or wild-type plants grown in the same conditions. The degree of complementation may vary, depending on the growth conditions, as shown during complementation analysis of the hy5 mutation using N-terminal TAPa-HY5 transgenic lines [7]. Thus, plants expressing TAPa-HY5 fully complemented the mutant background when grown under continuous red or blue light. However, the same transgenic lines grown under continuous far red light showed a partial complementation. For fusion protein purification purposes, proteins should be preferentially extracted from transgenic plants grown in the specific conditions where fully complementation or clear gain-of function phenotypes are observed. Additionally, fusions function can be assessed analyzing any biochemical or biophysical activity inherent to the target protein. As an example, functionality of CSN3 has been analyzed by testing the capacity of the CSN3-TAPa containing-CSN to derubylate cullin 1 in vitro and in vivo [7, 8]. Finally, if possible, gel filtration assays should be performed to determine whether TAPa fusions are correctly incorporated into endogenous protein complexes. In this regard, the gel filtration profile of the TAP fusion should be similar to that of the endogenous target protein, although a size difference between their peak fractions may occur, possibly due to the presence of the TAPa tag.

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