Molecular cloning and characterization of a group II chaperonin delta-subunit from soybean.
Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive. By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a delta-subunit homologue of CCT (CCTdelta). This subunit is responsible for the binding of an in vivo substrate, alpha-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear. As the cloning strategy, a putative amino acid segment, NH(2)-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCTdelta subunits, was chosen for designing a degenerate primer of the PCR-cloning. The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58-65% identity overall at the amino acid level with the corresponding subunits known to date. Using antibodies raised against Escherichia coli-produced soybean insoluble CCTdelta as a monitoring tool, we purified soybean CCT from the extract of its immature seeds. STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperonins. The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system. Northern blot analysis showed that soybean CCTdelta gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean.
Nong
Van Hai
VH
Genetic Engineering Laboratory, National Food Research Institute, Ministry of Agriculture, Forestry And Fisheries, Tsukuba Science City, Ibaraki 305-8642, Japan.
Arahira
Masaomi
M
Phan
Van Chi
VC
Kim
Chan-Shick
CS
Zhang
Deyu
D
Udaka
Kyoko
K
Fukazawa
Chikafusa
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D016252:Microscopy, Scanning Tunneling; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000737:chemistry; D013025:Glycine max / Q000235:genetics / Q000378:metabolism*
D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C533315:Tcp1 protein, mouse; D008156:Luciferases; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins