Expression, crystallization and preliminary crystallographic data analysis of filamin A repeats 14-16

Abstract

Human filamin A is a 280 kDa protein involved in actin-filament cross-linking. It is structurally divided into an actin-binding headpiece (ABD) and a rod domain containing 24 immunoglobulin-like (Ig) repeats. A fragment of human filamin A (Ig repeats 14-16) was cloned and expressed in Escherichia coli and the purified protein was crystallized in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. The crystals diffracted to 1.95 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.63, b = 52.10, c = 98.46 A, alpha = beta = gamma = 90 degrees.

Figure 1

SDS–PAGE showing tag-cleavage and gel-filtration fractions of human filamin A repeats 14–16. (a) Cleaved protein (lane 1), uncleaved protein (lane 2) and molecular-weight standards (lane 3) with sizes labelled in kDa. (b) Fractions from gel filtration (lanes 1–6) showing the 36.3 kDa protein and molecular-weight standards (lane 7).

Figure 2

Crystal (dimensions 0.3 × 0.2 × 0.05 mm) of filamin A repeats 14–16 in 1.6 M ammonium sulfate, 2% PEG 1000 and 100 mM HEPES pH 7.5. Alhough lacking distinct edges, this lens-shaped crystal diffracted to 1.7 Å.