Expression in Escherichia coli of human lamins A and C: influence of head and tail domains on assembly properties and paracrystal formation.
We have expressed in Escherichia coli cDNA corresponding to human lamins A and C, together with a number of fragments produced using site-specific mutagenesis. The proteins produced in this way were characterised both biochemically and ultrastructurally, and appeared to retain their native conformation. Crosslinking showed that all fragments formed 4-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 their proteolytically prepared rod domains. Moreover, the expressed lamins and a series of fragments in which different domains had been deleted formed paracrystals similar to those observed with native material. Deletion of either the N- or C-terminal non-helical domains altered the solubility and aggregation properties of the expressed protein, indicating that both domains have a role in lamin assembly.
Moir
R D
RD
MRC Laboratory of Molecular Biology, Cambridge, UK.
Donaldson
A D
AD
Stewart
M
M
D003001:Cloning, Molecular; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D034882:Lamins; D008854:Microscopy, Electron; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D012995:Solubility
D034882:Lamins; D009687:Nuclear Proteins