Protein destabilization using a conditional degron-based system
CM Hechenberger, Klaus Fortschegger, Sabine Strehl · Klinische Pädiatrie · 2017
Introduction: The most common ways to reduce the expression of a specific protein are to disrupt the encoding gene at the DNA level or to manipulate mRNA levels by RNA interference. As an alternative, tagging with an FK506- and rapamycin-binding protein (FKBP) based destabilization domain (DD) confers instability to the tagged protein in the absence of the compound Shield-1 allowing conditional and reversible protein depletion. We tested the feasibility of this approach to modulate the levels of the nuclear protein PAX5. Methods: We cloned four different constructs encoding human V5-PAX5 additionally tagged with N- or C-terminal single (1xDD) or double (2xDD) degron domains. The DD-tagged proteins were stably expressed in the B-cell leukemia cell line NALM-6 and the cells were cultivated with or without Shield-1. Protein levels were determined by Western blot analysis using a V5 antibody. Results: While a single degron domain was less sufficient for destabilizing PAX5, in the absence of Shield-1, both the N- and C-terminal 2xDD-tagged PAX5 proteins were decreased several-fold. Conclusion: Our results show that degron-tagging is a feasible approach to conditionally alter the protein level of the transcription factor PAX5.