Journal of Cell Science, Vol 93, Issue 1 71-83, Copyright © 1989 by Company of Biologists

JOURNAL ARTICLES

Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation

RA Quinlan, RD Moir and M Stewart
MRC Laboratory of Molecular Biology, Cambridge, England.

We have expressed in Escherichia coli a 1258 bp cDNA fragment corresponding to 97% of mouse glial fibrillary acidic protein (GFAP), the principal intermediate filament protein of astrocytes. High levels of expression were obtained, as a fusion protein with 32 residues of the bacteriophage lambda cII protein, using the pLcII expression vector system of K. Nagai and H.-C. Thogersen. Although removal of the cII protein fragment by proteolysis using factor X proved difficult, a protein corresponding to most of the cDNA fragment was obtained by cleaving at the endogenous thrombin site near the middle of the N-terminal non-helical domain of GFAP. A shorter 1047 bp fragment, in which the C-terminal non-helical domain of GFAP was deleted, was also produced using oligonucleotide-directed site-specific mutagenesis of the original cDNA clone. After proteolysis with thrombin, this material gave a fragment that corresponded to the alpha-helical coiled-coil rod region of the GFAP molecule, together with a portion of the non-helical N-terminal domain. The fragments produced were characterized both biochemically and ultrastructurally, and appeared to retain the conformation of native GFAP. Crosslinking showed that all fragments formed molecules containing two chains ('dimers') that associated to form four-chain molecular dimers ('tetramers') analogous to those formed by intact intermediate filament proteins. Shadowed preparations showed the presence of rod-like particles that closely resembled those observed for other intermediate filament proteins and proteolytically prepared rod domains. Remarkably, the fusion protein produced from the entire 1258 bp cDNA fragment and the cII peptide was able to form filaments that closely resembled those produced by native GFAP. However, fragments in which either the cII peptide or the C-terminal non-helical domain were removed, or in which both were removed, failed to form filaments under standard assembly conditions. Although preliminary in nature, these results suggest that both N- and C-terminal non-helical domains may have a role in intermediate filament formation. Moreover, the fragment corresponding approximately to the GFAP rod formed paracrystals similar to those observed with other coiled-coil proteins. The molecules in these paracrystals were arranged antiparallel with the two molecules in the unit cell, which may correspond to the four-chain molecular dimer (tetramer), overlapping by approximately two-thirds of their length.
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