Yeast heat shock protein 60 and analogs

Details Yeast heat shock protein 60 and analogs Yeast heat shock protein 60 and analogs

1991-03-18

2001-04-10

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S252300, C435S320100, C435S325000, C536S023100, C536S023700

Reexamination Certificate

active

06214606

ABSTRACT:

DESCRIPTION
1. Technical Field
The present invention relates to yeast heat shock protein 60 (hsp60) and analogs thereof that are useful in assembling biologically active proteins.
2. Background of the Invention
The translocation of proteins through biological membranes is a phenomenon of fundamental importance, enabling the compartmentation of eurcaryotic cells. Recent studies of protein translocation have focused on determination of the tertiary structure of proteins that are translocated, and on identification of mediating components. Particular attention has been directed to mitochondria because most of the proteins residing inside these organelles are initially synthesized outside, in the cytosol, and then translocated to the innermost matrix compartment.
Several studies have indicated that newly-synthesized mitochondrial precursor proteins must assume an “unfolded” conformation in order to be translocated, and a recent analysis suggests that this translocation-competent conformation may be directed by a family of hsp70 proteins in the cytosol. After passage through the mitochondrial membranes at points of contact between outer and inner membranes, and following subsequent proteolytic removal of NH
2
-terminal signal peptides, mitochondrial subunits fold or assemble (fold/assemble) into their biologically active conformations. This step might not occur spontaneously but rather it might be an active step, requiring the action of one or more gene products. If such products exist, the gene products may be necessary to produce functional conformations of biologically active molecules which are chemically synthesized or produced recombinantly in a host cell lacking the gene product.
Hemmingsen et al. [
Nature
, 333:330-334 (1988)] describe a family of proteins they term “molecular chaperones” which are associated with post-translational assembly of proteins. The proposed role of the chaperones is to ensure that the folding of certain polypeptides and their assembly into oligomeric structures occurs correctly, presumably by preventing formation of “improper” structures resulting from the exposure of hydrophobic or charged surfaces either within or between polypeptide chains. The authors state that the three classes recognized within the molecular chaperone family are (1) nucleoplasmin, (2) hsp70-immunoglobulin heavy chain binding protein class, and (3) the bacterial-mitochondrial-chloroplast class. The authors propose the term “chaperoning” to apply to the third class.
Hemmingsen et al. suggest that those biotechnologists encountering problems or producing foreign proteins in bacterial cells may be experiencing a failure of a bacterial chaperonin to be able to mediate folding/assembly normally performed by a chloroplast or mitochondrial chaperonin.
Determining those proteins necessary for proper folding/assembly of foreign proteins in a host cell would facilitate production of active, biologically useful proteins.
BRIEF SUMMARY OF THE INVENTION
The present invention has several facets. Those facets revolve around a yeast (
S. cerevisiae
) heat shock protein that plays a pivotal role in protein assembly and/or activation, particularly for mitochondrial proteins.
In one aspect, the invention contemplates a purified, biologically active protein having an M
r
of about 55,000 to about 65,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. That protein exhibits at least about 60 percent amino acid residue identity to the yeast mitochondrial heat shock protein whose amino acid residue sequence is shown in
FIG. 1
from amino acid residue position 23 through position 572. That yeast protein is referred to herein as hsp60 or an analog thereof.
The invention further contemplates a DNA segment consisting essentially of an isolated, non-chromosomal DNA segment containing sufficient DNA to encode hsp60 or an analog. The DNA segment typically contains about 1600 to about 2300 base pairs, and preferably encodes the amino acid residue sequence shown in
FIG. 1
from nucleotide position 67 through nucleotide position 1716.
Another embodiment of the invention contemplates a vector capable of autonomous replication in a cell. The vector contains an operatively linked polynucleotide sequence segment that encodes hsp60 or an analog thereof. The vector is preferably a DNA vector that directs expression of hsp60 or its analog.
Still another embodiment contemplates a transformed host cell containing an above-described vector. That host cell can be eucaryotic or procaryotic.
Yet other embodiments of the invention relate to methods.
In a first method, a functional oligomeric protein complex is formed. Here, non-functional protein subunits are admixed in vitro with a functionalizingly effective amount of a biologically operative aqueous yeast mitochondrial matrix preparation to form an aqueous admixture. That matrix preparation contains at least about twice the amount of hsp60 or an analog than the amount of hsp60 present after heat shock up to about 10 percent of the total protein in the matrix preparation. The admixture is maintained under biological culture conditions for a time period sufficient for the non-functional protein subunits to bind to hsp60 or analog, be processed and folded, and to dissociate therefrom to form a functional oligomeric protein complex.
In a second method, an inactive form of monomeric protein molecules or protein subunit molecules is converted to an active form. In this method, inactive monomeric protein molecules or protein subunit molecules are admixed in vitro with an activatingly effective amount of a biologically operative, aqueous yeast mitochondrial matrix preparation as discussed before, to form an aqueous admixture. The admixture is maintained under biological culture conditions for a time period sufficient for the inactive monomeric protein molecules or inactive protein subunits to bind to hsp60 or its analog, be processed and folded, and to dissociate to form an active form of the monomeric protein molecules or protein subunit molecules.
Third and fourth methods are substantially identical to the above first and second methods, respectively, except that the mitochondrial matrix preparation is used as is and does not include added hsp60 or an analog protein molecule.
In each of the above-discussed methods, the non-functional protein subunits, or inactive monomeric protein molecules or inactive protein subunit molecules are typically present at a weight ratio relative to the total protein of the matrix preparation of about 1 to about 20 parts relative to about 200 to about 2000 parts, respectively. The total protein concentration of an aqueous admixture is about 10 to about 70 milligrams per milliliter.


REFERENCES:
Jindal et al.,Mol. Cell. Biol., 9(5):2279-2283 (1989).
Mehra et al.,Proc. Natl. Acad. Sci. USA, 83:7013-7017 (1986).
Sigma Chemical Co. catalogue Feb. 1986; pp. 844, 845 & 849.*
Ingolia et al., Molecular & Cellular Biology 2 (11) 1388-98 (1982).*
Moran et al., Can J Biochem Cell Biol 61 (6) 488-499 (1983).*
Hickey et al., Gene 43 147-154 (1986).

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