Expression, purification, crystallization and X-ray diffraction studies of the molecular chaperone prefoldin from Homo sapiens

Abstract

Proper protein folding is an essential process for all organisms. Prefoldin (PFD) is a molecular chaperone that assists protein folding by delivering non-native proteins to group II chaperonin. A heterohexamer of eukaryotic PFD has been shown to specifically recognize and deliver non-native actin and tubulin to chaperonin-containing TCP-1 (CCT), but the mechanism of specific recognition is still unclear. To determine its crystal structure, recombinant human PFD was reconstituted, purified and crystallized. X-ray diffraction data were collected to 4.7 Å resolution. The crystals belonged to space group P21212, with unit-cell parameters a = 123.2, b = 152.4, c = 105.9 Å.

Keywords: molecular chaperone; prefoldin; protein folding.

Figure 1

Biochemical characterization of human PFD. (a) SDS–PAGE analysis of purified human PFD. Lane M, molecular-weight marker (labelled in kDa); lane 1, peak fraction from Superdex 200 column. PFD subunits are assigned to the bands according to the molecular weights calculated from the sequences. (b) SEC-MALS of purified PFD. Red, molecular mass; blue, absorbance at 280 nm. (c) CD spectra. Red, spectrum of human PFD (HsPFD); blue, spectrum of P. horikoshii PFD (PhPFD). (d) MALDI-TOF MS. Peaks are annotated with the observed molecular mass (black) and the calculated mass from the sequence (blue, in parentheses).

Figure 2

Crystals of human PFD. The black bar represents 0.5 mm.

Figure 3

X-ray diffraction pattern from a crystal of human PFD. The circle represents 4.7 Å resolution.