Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-05-30
2004-02-17
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S252300, C435S325000, C536S023100, C530S350000
Reexamination Certificate
active
06692935
ABSTRACT:
BACKGROUND OF THE INVENTION
Increasingly, there is a need for proteins which combine two or more functions, such as binding or catalysis, in a single structure. Typically, proteins which combine two or more functions are prepared either as fusion proteins or through chemical conjugation of the component functional domains. Both of these approaches suffer from disadvantages. Genetic “single chain” fusions suffer the disadvantages that (i) only a few (2-3) proteins can be fused (Rock et al., 1992
, Prot. Eng.
5, 583-591), (ii) mutual interference between the component domains may hinder folding, and (iii) the size of the fusion protein may make it difficult to prepare. The alternative, chemical cross-linking in vitro following purification of independently expressed proteins, is difficult to control and invariably leads to undefined products and to a severe loss in yield of functional material.
Recently, methods for achieving non-covalent association of two or more of the same functional domains have been developed. This can be achieved through the use of domains attached to peptides which self-associate to form homo-multimers (Pack & Plückthun, 1992,
Biochemistry
31, 1579-1584). For example, the association of two separately expressed scFv antibody fragments by C-terminally fused amphipathic helices in vivo provides homo-dimers of antibody fragments in
E. coli
(PCT/EP93/00082; Pack et al., 1993,
Bio/Technology
11, 1271-1277) or homo-tetramers; (Pack et al., 1995,
J. Mol. Biol.,
246, 28-34).
To assemble distinct protein functions such as two antibody fragments with different specificities fused to such association domains, the helices must have a tendency to form hetero-multimers. In principle, this could be achieved with complementary helices such as the hetero-dimerizing JUN and FOS zippers of the AP-1 transcription factor (O'Shea et al, 1992,
Cell
68, 699-708). The clear disadvantage of association domains based on hetero-associated helices, however, is their pseudo-symmetry and their similar periodicity of hydrophobic and hydrophilic residues. This structural similarity results in a strong tendency to form homo-dimers and, thus, to lower significantly the yield of hetero-dimers (O'Shea et al., 1993,
Cell
68, 699-708; Pack, 1994, Ph. D. thesis, Ludwig-Maximilians-Universität München). Furthermore, the formation of JUN/FOS hetero-dimers is kinetically disfavoured and requires a temperature-dependent unfolding of the kinetically favoured homo-dimers, especially JUN/JUN homo-dimers (PCT/EP93/00082; O'Shea et al., 1992,
Cell
68, 699-708; Pack, 1994, Ph. D. thesis, Ludwig-Maximilians-Universität München). Because of the need of additional purification steps to separate the unwanted homo-dimers from hetero-dimers and the resulting decrease in yield, hetero-association domains based on amphipathic helices do not result in practical advantages compared to conventional chemical coupling.
These disadvantages of the prior art are overcome by the present invention which provides multi-functional polypeptides and methods for the preparation of these multi-functional proteins. This is achieved via the use of association domains which are designed to associate predominantly in a complementary fashion, and not to self-associate.
DETAILED DESCRIPTION OF THE INVENTION
In the earliest steps of protein folding, peptide chains form a disordered hydrophobic core by collapsing hydrophobic residues into the interior of an intermediate “molten globule”. This hydrophobic effect is considered to be the most important driving force of folding (Matthews, 1993,
Annu. Rev. Biochem.
62, 652-683; Fersht, 1993,
FEBS Letters
325, 5-16). The burial of hydrophobic residues and the resulting exclusion of solvent is the determining factor in the stability of compact tertiary structures such as acyl-phosphatase (Pastore et al, J. Mol. Biol. 224, 427-440, 1992) interleukin-2 (Brandhuber et al., 1987,
Science
238, 1707-1709), calbindin (Parmentier, 1990,
Adv. Exp. Med. Biol.
269, 27-34) or ubiquitin (Briggs & Roder, 1992,
Proc. Natl. Acad. Sci. USA
89, 2017-2021).
This concept forms the basis of the present invention, which provides individually encoded peptides or “segments” which, in a single continuous chain, would comprise a compact tertiary structure with a highly hydrophobic core. The component peptides are chosen so as to be asymmetric in their assumed structure, so as not to self-associate to form homo-multimers, but rather to associate in a complementary fashion, adopting a stable complex which resembles the parent tertiary structure. On the genetic level, these segments are encoded by interchangeable cassettes with suitable restriction sites. These standardized cassettes are fused C- or N-terminally to different recombinant proteins via a linker or hinge in a suitable expression vector system.
Thus, the present invention relates to a multi-functional polypeptide comprising:
(a) a first amino acid sequence attached to at least one functional domain;
(b) a second amino acid sequence attached to at least one further functional domain; and
(c) optionally, further amino acid sequences each attached to at least one further functional domain;
wherein any one or more of said amino acid sequences interacts with at least one of said amino acid sequences in a complementary fashion to form a parental, native-like tertiary or optionally quaternary structure and wherein the parental, native-like tertiary or optionally quaternary structure is derived from a single parent polypeptide. In this context, the term parent polypeptide refers to a polypeptide which has a compact tertiary or quarternary structure with a hydrophobic core. The invention provides for many different parent polypeptides to be used as the basis for the association domain. Suitable polypeptides can be identified by searching for compact, single-domain proteins or protein fragments in the database of known protein structures (Protein Data Bank, PDB) and selecting structures that are stable and can be expressed at high yields in recombinant form. These structures can then be analyzed for hydrophobic sub-clusters by the method of Karpeisky and Ilyn (1992,
J. Mol. Biol.
224, 629-638) or for structural units (such as &bgr;-elements or helical hairpin structures) by standard molecular modelling techniques. In a further embodiment, the present invention provides for multi-functional polypeptides wherein the single parent polypeptide is taken from the list ubiquitin, acyl phosphatase, IL-2, calbindin and myoglobin.
In a preferred embodiment, the present invention provides a multi-functional polypeptide comprising two or more amino acid sequences each attached to at least one functional domain, wherein any two or more of said amino acid sequences can associate in a complementary fashion to provide a parental, native like, tertiary or optionally quaternary structure.
The structure of the multifunctional complex of the present invention is described schematically in FIG.
1
. Polypeptide segments which do not have the ability to assemble as homodimers are derived by cutting a parental polypeptide which has a compact tertiary structure and a highly hydrophobic core. These polypeptide segments can then fused to one or more different functional domains at the genetic level. These distinct polypeptide segments which are now fused to one or more functional domains can be, for example, coexpressed resulting in the formation of a native like parental structure attached to functional domains. This parental structure is formed by the dimerization of the polypeptide segments which were derived from the original parental polypeptide. The resulting multifunctional complex, as pictured in
FIG. 1
, would appear as a compact tertiary structure attached to the one or more functional domains.
Once structural sub-domains are identified, the protein is dissected in such a way these sub-domains remain intact. The selection process can be expanded to proteins for which no structure is available but which satisfy the criteria of stability and
Lupas Andrei
Pack Peter
Carlson Karen Cochrane
Heller Ehrman White & McAuliffe LLP
MorphoSys AG
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