Expression of plant chaperonin-60 genes in Escherichia coli

• Published in: The Journal of biological chemistry Vol. 267; no. 32; pp. 23327 - 23332
• Main Authors: CLONEY, L. P, WU, H. B, HEMMINGSEN, S. M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 15.11.1992
• Subjects: Amino Acid Sequence; Antibodies; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Biological and medical sciences; Brassica - genetics; brassica napus; Chaperonin 60; chloroplaste; chloroplasts; Cloning, Molecular - methods; cloroplasto; escherichia; Escherichia coli - genetics; expresion genica; expression des genes; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Plant; Genetic Vectors; Heat-Shock Proteins - biosynthesis; Heat-Shock Proteins - genetics; Heat-Shock Proteins - isolation & purification; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Molecular Weight; Oligopeptides - chemical synthesis; Oligopeptides - immunology; peptide; peptides; peptidos; plant protein; plasmide; plasmidios; plasmids; Promoter Regions, Genetic; proteinas vegetales; proteine vegetale; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Escherichia coli; Gene expression; Transfection; Gene; Cruciferae; Dicotyledones; Angiospermae; Brassica napus; Immunoblotting assay; Bacteria; Spermatophyta; Chloroplast; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)50094-6

We have examined the expression in Escherichia coli of genes encoding a plant chloroplast molecular chaperone, chaperonin-60. Purified plant chaperonin-60 is distinct in that it contains two polypeptides, p60cpn-60 alpha and p60cpn-60 beta, which have divergent amino acid sequences (Hemmingsen, S. M., and Ellis, R. J. (1986) Plant Physiol. 80, 269-276; Martel, R., Cloney, L. P., Pelcher, L. E., and Hemmingsen, S. M. (1990) Gene (Amst.) 94, 181-187). The precise polypeptide composition(s) of the active tetradecameric specie(s) (cpn60(14)) has not been determined. Genes encoding the mature forms of the Brassica napus chaperonin polypeptides have been expressed separately and in combination in E. coli to produce three novel strains: alpha, beta, and alphabeta. The plant cpn60 polypeptides accumulated in soluble forms and to similar high levels in each. There was no conclusive evidence that p60cpn-60 alpha assembled into cpn60(14) species in alpha cells. In beta and alphabeta cells, the plant gene products assembled efficiently into cpn60(14), species. Thus, the assembly of p60cpn-60 alpha required the presence of p60cpn-60 beta, whereas the assembly of p60cpn-60 beta could occur in the absence of p60cpn-60 alpha. Significant proportions of the endogenous groEL polypeptides were not assembled into tetradecameric groEL14 in beta and alphabeta cells. Analysis of the tetradecameric species that did form indicated the presence of novel hybrid cpn60(14) species that contained both plant and bacterial cpn60 polypeptides.