Folding protein complexes

Details Folding protein complexes Folding protein complexes

1996-10-11

2001-07-03

Mertz, Prema (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S252300, C435S254110, C435S325000, C435S320100, C435S471000, C536S023500, C530S325000, C530S350000

Reexamination Certificate

active

06255070

ABSTRACT:

The present invention relates to the folding of proteins. In particular, the present invention provides proteins, which are able to form a complex in vitro, useful in facilitating folding of proteins, for example those produced using recombinant DNA technology. Genes encoding the proteins of the complex are also provided. The present invention further relates to methods of assembling a protein complex able to fold proteins in an in vitro environment.
Molecular chaperones are known to be able to assist in the folding of proteins along the folding pathways from denatured state to correctly folded product (reviewed in [1]). One well studied class of molecular chaperones are the chaperonins (GroEL, Hsp60 and Rubisco subunit binding protein) found in eubacteria, mitochondria and plastids (reviewed in [2,3]). They are 14-mer double-torus structures composed of one (GroEL and Hsp60) or two (Rubisco subunit binding protein) subunit types and show seven-fold symmetry [4,5]. In vitro these chaperonins bind denatured proteins and upon ATP hydrolysis, release them into aqueous solution where they complete folding [6]. In vivo there is evidence that they are involved in the folding, transport and assembly of newly synthesized proteins. The original mutations isolated in groE affected the folding of bacteriophage particle subunits [7] but more recent genetic analysis suggests a more general role in protein biogenesis in
E. coli [
8]. HSD60 is involved in the import of proteins into the mitochondrial lumen from the cytoplasm [9].
Although no GroEL-like chaperonins have been identified in eukaryotic cytosol, the double-torus TCP-1-containing particle seems to be a component of the eukaryotic folding machinery and may play an analogous role to that of GroEL in eubacteria and the GroEL related chaperonins in symbiotic organelles. TCP-1 is weakly related to the GroEL family [10] but shows nearly 40% identity to an archaebacterial chaperonin, TF55 [11]. It has been proposed that GroEL and TCP-1 are subfamilies derived from a primordial gene [10,12,13,14] and it has been suggested that the eukaryotic TCP-1-containing chaperonin may have evolved from an archaebacterial lineage [3,11].
Recently, purified chaperonin containing TCP-1 has been shown to facilitate the folding of actin [15] and tubulin [16,17] in vitro and it binds newly-synthesized actin, tubulin and some other unidentified polypeptides in vivo [18]. One striking difference between the bacterially derived chaperonins and the TCP-1-containing chaperonin is the heteromeric nature of the TCP-1-containing particle [14,15,17,19]. There are at least five polypeptide species in the complex containing TCP-1 [14,17].
To date, little sequence information has been available on the polypeptides which make up the complex, despite the fact that various parties have obtained the sequences of peptides from a number of polypeptides of TCP complexes of different organisms. Frydman et al (17) demonstrated the presence of six subunits in bovine TCP complex, which they termed “TRiC” (TCP-1 ring complex), and obtained some peptide sequence information indicating some resemblance both between polypeptides of the complex and between these polypeptides and those of other organisms. Rommelaere et al [59] looked at the cytosolic chaperonin from both rabbit reticulocyte lysate and bovine testis. They report finding eight different polypeptides in rabbit reticulocyte chaperonin, and obtaining partial amino acid sequences of all eight.
However, full length clones have proved elusive. The full sequence of murine TCP-1 has been available since 1986 (20) and Ehmann et: al (FEBS, 336: 2, 313-316, 1993) have reported the obtention of a TCP-1 related sequence from
Avena sativa
(oat) seedlings. Despite this information being available, there has yet to be a report of the obtention of full-length nucleic acid sequences encoding the components of a mammalian TCP-1 complex. Knowledge of short peptide sequences derived from individual subunits of chaperonin containing TCP-1 has not enabled the specific cloning of the full-length cDNA for individual subunits. One problem is that in order to be sure a peptide sequence is derived from a subunit of the family, it must be identifiably homologous to the only full-length mammalian sequence available, ie TCP-1 (20,48). Any DNA sequences derived by reverse translation from the novel peptide sequence will also be related to TCP-1 and the related gene sequences. If these sequences are used as PCR primers they will prime synthesis and amplification of many TCP-1 related sequences, so further insight and activity are needed to identify the sequences which encode particular subunits of the complex.
The present invention provides individually seven nucleic acid molecules with sequences encoding subunits of the TCP-1-containing chaperonin, different from the original Tcp-1 gene (reported in 20). Since, in mice, at least three of the novel Tcp-1 related genes are unlinked to the mouse t complex, it is proposed to rename the TCP-1 complex [14] as CCT,
c
haperonin
c
ontaining
T
CP-1. Only now we have all the eight complete sequences of the ubiquitously expressed subunits is it possible to know the gene and subunit to which each PCR product corresponds. Likewise, all other TCP-1 related genes in the databases make no sense without the complete sequences being available.
The present invention also provides molecules which are mutants, derivatives or alleles of any one of the seven sequences provided, particularly mutants, derivatives and alleles which encode a protein which retains a functional characteristic of the protein encoded by the respective wild-type gene, especially the ability to associate with at least another subunit to form a complex able to fold a polypeptide. Changes to a sequence, to produce a mutant or derivative, may be by one or more of insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the insertion, deletion or substitution of one or more amino acids. Of course, changes to the nucleic acid which make no difference to the encoded amino acid sequence are included. We have demonstrated the existence of the 8 gene sequences in yeast and in plants by hybridization (52, unpublished results) and 6 genes have been isolated from yeast by ourselves and others (53). These 6 yeast genes correspond exactly (ie. they are the orthologues of) to six of the genes exemplified in this application CCT&agr;, &bgr;, &ggr;, &dgr;, &egr;, &eegr; and &zgr;. We predict that all eukaryotic organisms contain at least the set of eight genes which we have described in mouse. There may be tissue specific CCT genes or additional CCT genes in some organisms but we would expect each of these to be closely related (greater than 70% amino acid sequence homology) to one of the eight genes described here. These eight CCT genes of mouse constitute the basic family which comprise the core CCT complex. In a preferred embodiment of the present invention, the sequence is one encoding a polypeptide found in a human or a mouse.
The polypeptides may have an amino acid sequence which shares a significant degree of homology with any of the specific sequences provided herein. Such homology may for example, be 60% or greater, 70% or greater, 80% or greater, 90% or greater or 95% or greater, provided the polypeptide is able to function as a subunit of a complex able to fold a polypeptide
The sequences of polypeptides encoded by nucleic acid according to each of seven different embodiments of the present invention are provided in FIGS.
3
(
a
) to (
f
) and FIG.
8
(
h
) Preferred nucleic acid sequences are shown in FIGS.
8
(
b
) to (
h
). The present invention also provides a vector which comprises nucleic acid with any one of the provided sequences, preferably a vector from which polypeptide encoded by the nucleic acid sequence can be expressed. T

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