Analysis of cytokeratin domains by cloning and expression of intact and deleted polypeptides in Escherichia coli.
Using recombination of an appropriate expression vector system (pINDU) with a complete cDNA encoding a basic (type II) cytokeratin, i.e. cytokeratin 8 (1) of Xenopus laevis, we transformed Escherichia coli cells to synthesize considerable amounts of an insoluble eukaryotic cytoskeletal protein. The cytokeratin was deposited in large 'inclusion bodies' in the bacterial cytoplasm but did not form detectable filamentous structures. However, when the E. coli-expressed cytokeratin was purified and combined in vitro with an authentic cytokeratin of the complementary, i.e. acidic (type I) subfamily, it formed typical intermediate-sized filaments (IFs). Using Bal31 deletion from either the 5' or the 3' end of the cDNA, series of polypeptides progressively deleted from the amino or the carboxy terminus were produced in E. coli and identified by monoclonal antibodies. These assays allowed the mapping of epitopes. The deletion polypeptides of cytokeratin 8 were further examined to localize the region(s) involved in the heterotypic binding of alpha-helices of type I cytokeratins, using an in vitro nitrocellulose blot binding assay. We show that a region of 37 amino acids located in the central portion of coil 2 of the alpha-helical rod domain is sufficient for the specific recognition of a radiolabelled type I cytokeratin, i.e. cytokeratin 18 (D) from rat liver. In addition, deletion polypeptides containing only coil 1 of the alpha-helical rod also bind strongly the complementary cytokeratin. This indicates that the capability of heterotypic recognition and complex formation is not restricted to a single signal sequence but is located in distant and independent alpha-helical domains.(ABSTRACT TRUNCATED AT 250 WORDS)
D000595:Amino Acid Sequence; D000818:Animals; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005822:Genetic Vectors; D007633:Keratins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification; D014158:Transcription, Genetic; D014982:Xenopus laevis
D011994:Recombinant Proteins; D007633:Keratins; D004247:DNA