Development of a method for screening short-lived proteins using green fluorescent protein

Abstract

We have developed a screening technology for the identification of short-lived proteins. A green fluorescent protein (GFP)-fusion cDNA library was generated for monitoring degradation kinetics. Cells expressing a subset of the GFP-cDNA expression library were screened to recover those in which the fluorescence signal diminished rapidly when protein synthesis was inhibited. Thirty clones that met the screening criteria were characterized individually. Twenty-three (73%) proved to have a half-life of 4 hours or less.

Figure 1

Schematic diagram of the four steps of the screening procedure. (a) Fractionate by FACS cells transfected with an EGFP-cDNA expression library according to their fluorescence intensities; (b) refractionate those cells made dimmer by cycloheximide (CHX) treatment; (c) recover plasmids, clone in bacteria, pool clones and select CHX-responsive pools by FACS analysis; (d) recover and characterize individual cDNA clones.

Figure 2

Fractionation of 293T cells transfected with a GFP-cDNA expression library. Cells were subjected to FACS analysis and fractionated into five subpopulations: R2, R3, R4, R5 and R6.

Figure 3

FACS analysis of fractionated cells treated with CHX or untreated. The fractionated subpopulations R3 and R4 treated with or without CHX were subjected to FACS analysis. The log-normal fluorescence histogram distributions from (a) R3 and (b) R4 populations are shown. The gray curve represents cell populations not treated with CHX and the black curve represents the treated cells. The shaded area represents cells from the populations left-shifted by CHX that were used for plasmid recovery.

Figure 4

CHX chase analysis by western blot of three labile EGFP-fused library clones. (a) Cells were individually transfected with GFP-cDNA clones representing splicing factor SRp30c, guanine nucleotide-binding regulatory protein or cervical cancer proto-oncogene p40. Transfected cells were treated with CHX and were collected immediately thereafter or after 1, 2 or 3 hours for western blot analysis using anti-EGFP polyclonal antibody. The mobility of protein markers is indicated. (b) Cells were transfected with constructs expressing EGFP or d1EGFP, a destabilized form of GFP, and analyzed as in (a).

Figure 5

Cycloheximide chase analysis by western blot of three full-length myc-tagged cDNAs. Cells were transiently transfected to express splicing factor SRp30c, guanine nucleotide-binding regulatory protein or cervical cancer proto-oncogene p40, each with an amino-terminal myc epitope tag. Transfected cells were treated with CHX and samples subjected to western blot analysis using anti-myc antibody. The mobility of protein markers is indicated.

Figure 6

Cycloheximide chase analysis by western blot of two endogenous proteins. 293T cells were treated with CHX and samples subjected to western blot analysis using antibodies against G protein or Hsp70. The mobility of protein markers is indicated.