Induction of extremely low protein expression level by fusion of C‐terminal region of Nef

Abstract

Nef is one of the accessory proteins of human immunodeficiency viruses. Here, we noted that the relative expression level of Nef(NL4-3) is much lower than that of NefJR-CSF in HEK293 cells. By evaluating the expression level using a Nef mutant, it was indicated that amino acids 129-206 of Nef(NL4-3), that is, the C-terminal region named NLAA129-206, could contain the region responsible for the induction of the low protein expression level. In addition, the expression levels of the enhanced green fluorescent protein and Renilla luciferase became extremely low with the fusion of NLAA129-206. Interestingly, the NLAA129-206-corresponding sequences of other Nef variants with relatively high expression levels also induced the extremely low protein expression level by fusion. These results suggest that the C-terminal region of Nef can generally induce an extremely low protein expression level. Here, we propose that the C-terminal region of Nef could become an excellent tool for the induction of an extremely low expression level of arbitrary proteins by attachment as fusion proteins.

Clearly different mechanisms of enhancement of short-lived Nef-mediated viral infectivity between SIV and HIV-1

“…These signals induce a rapid protein degradation mediated by a proteasome, in which the CL peptide requires ubiquitination prior to degradation, whereas the PEST sequence requires no ubiqutination [ 14 ]. The CL peptide and PEST sequence convert stable proteins into unstable ones by attachment as fusion proteins [ 28 , 30 - 32 ], whose apparent expression levels could be much lower than those of the original proteins [ 30 - 33 ]. In this study, we utilized a combination of two protein degradation sequences of the CL peptide and PEST sequence, namely, the CP sequence, to generate short-lived Nef.…”