Expression of plant chaperonin-60 genes in Escherichia coli.
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 alpha beta. 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 alpha beta 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 alpha beta cells. Analysis of the tetradecameric species that did form indicated the presence of novel hybrid cpn6014 species that contained both plant and bacterial cpn60 polypeptides.
Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan.
L P
LP
Cloney
H B
HB
Wu
S M
SM
Hemmingsen
D000595:Amino Acid Sequence; D000906:Antibodies; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001937:Brassica / Q000235:genetics*; D018834:Chaperonin 60; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D017343:Genes, Plant; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D000906:Antibodies; D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins