Trimerizing polypeptides, their manufacture and use

Details Trimerizing polypeptides, their manufacture and use Trimerizing polypeptides, their manufacture and use

1997-01-10

2001-02-20

Romeo, David (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...

C435S325000, C435S252300, C435S320100, C530S324000, C530S356000, C530S387300, C530S402000, C536S023400

Reexamination Certificate

active

06190886

ABSTRACT:

The present invention relates to polypeptides able to form multimers, particularly trimers, and manufacture and use of such polypeptides.
The biosynthesis of collagen molecules requires the correct alignment of three polypeptides consisting of Gly-Xaa-Yaa triplets to form the triple-helix [1]. Each chain assumes a left-handed helical structure in the right-handed triple-helix, which is stabilized by inter-chain hydrogen bonds. The formation of the triple helix proceeds from a single nucleation point at the C-terminal end of the three chains and grows in a zipper-like fashion [2].
Refolding experiments on collagen type III indicated that specific inter-chain disulphide-bridges formed between C-terminal globular protein structures, can be sufficient to function as a nucleus for the refolding of a triple-helix in vitro, whereas reduction abrogates this process completely [3]. However, the molecular mechanism guiding association and registered alignment of collagens has remained elusive since the family of proteins containing collagenous sequences is large and sequence comparison of the different types of C-terminal, non-collagen-like, regions did not reveal a common motif shared by FACITs (fibril associated collagens with interrupted triple-helix, types IX, XII, XIV, and XVI), the collagens of striated fibrils (types I, II, III, V, and XI), or the collagens with Clq-like C-terminal domains (types VIII and X) [4]. The frequent formation of inter-chain disulphide bonds has further complicated the search for protein modules involved in the inter-chain association and subsequent nucleation of triple-helix formation.
The family of collagenous proteins known as the “collectins” is composed of the serum proteins mannan-binding protein (MBP), collectin-43 and bovine conglutinin as well as the lung surfactant proteins SP-D and SP-A [5]. Collectin polypeptide chains contain a short N-terminal region, a collagen-like region (of between 20 and 59 Gly-Xaa-Yaa triplets) linked, by a short stretch of 34-39 amino acids (which form the ‘neck’ region) to a C-terminal, C-type lectin domain (of 113-118 amino acids) (
FIG. 1
a
).
The present invention has resulted from results showing that the “neck-region” of collectin protein is able to mediate inter-chain recognition, trimerization and registered alignment of three collagenous polypeptide chains [7]. The results make available simple means of trimerising polypeptides of choice.
According to the present invention there is provided a polypeptide comprising a neck-region of a collectin, or an amino acid sequence variant thereof or a derivative thereof. Such polypeptide will form a trimer under appropriate conditions. The polypeptide is non-naturally occurring, i.e. it is one not found in nature.
It may comprise one or more heterologous amino acids joined to the neck-region or variant or derivative thereof. It may retain one or more amino acids from the molecule from which is it derived; for example the polypeptide may comprise a collectin C-type lectin domain.
According to one definition, the present invention provides a non-naturally occurring polypeptide consisting essentially of amino acids according to the following formula:
X—N—Y,
wherein N is a collectin neck-region peptide or a variant or derivative thereof or a sequence of amino acids having an amino acid pattern and/or hydrophobicity profile which is the same as or similar to that of a collecting neck-region, able to form a trimer; X is absent or one or more amino acids and Y is absent or one or more amino acids. If X and Y are both absent, the polypeptide consists essentially of N. X and/or Y may comprise one or more heterologous amino acids, any of which may be derivatisable, or “chemically modifiable”, for attachment of a chemical moiety.
The chemical moiety may be introduced at a specific chemically modifiable residue or residues. A chemically modifiable amino acid residue is an amino acid residue susceptible to modification with a chosen chemical reagent under specified conditions. The amino acid may be unique in the polypeptide or it may be uniquely modifiable, or selectively or preferentially modifiable over other amino acids present. For instance, a cysteine residue may be introduced into the binding site and be available for chemical modification via its thiol group. It may also be possible to render an amino acid preferentially modifiable compared with other amino acids of the same type within the molecule by engineering its environment, eg by positioning it within the molecule adjacent another amino acid with particular properties. For instance, an amino group next to a carboxlate group would be rendered more nucleophilic and selectively modifiable even if not unique within the binding site.
Other chemically modifiable amino acids include lysine, glutamate, histidine and tyrosine.
Covalent modification allows a wide variety of moieties to be incorporated, particularly reporter groups or cofactors for catalysis. In one embodiment of the present invention, one or more amino acids which are specifically modifiable are incorporated. This allows the interaction of large organic groups such as the fluorescent reporter group, 7-nitrobenz-2-oxa-1,3-diazole (NBD). Other large groups such as the flavin cofactors for catalysis, FMN and FAD may be incorporated.
There is also the possibility of incorporating two (or more) residues for modification with the same reagent or two (or more) different reagents, or more preferably different residues may be modified with different reagents to incorporate different chemical moieties into the binding site. This is useful eg for catalysis where the presence of two chemical moieties such as flavin and haem may promote catalysis of a redox reaction.
There are other possible ways of modifying a polypeptide. There are a number of amino acid residues which may be specifically derivatized using molecules containing specific functional groups. For instance, amino groups may be modified with N-hydroxysuccinimide esters, carboxyl groups with carbodiimides, histidines and cysteines with halomethyl ketones, arginine with glyoxals (see e.g. A. R. Fersht, Enzyme Structure and Mechanism 2nd edn, 1985 pp248-251, W. H. Freeman, New York).
Some reagents which may be used to modify specific amino-acid residues are given by T. Imoto and H. Yamada in “Protein Function: a Practical Approach”, pp247-277, 1989. To introduce specific functional groups into polypeptides the reactive group of these reagents may be combined with the functional group in a modifying reagent. For instance, if it is desired to modify a protein with the fluorophore 7-amino-4-methylcoumarin-3-acetic acid, the N-hydroxysuccinimidyl ester of the molecule may be used to modify amino groups, whereas N-[6-(-amino-4-methylcoumarin-3-acetamido)hexyl]-3′-(2′-pyridyldithio)propionamide may be used to modify cysteine groups.
Another possible methodology is to use transglutaminase which catalyzes an acyl-transfer reaction between the gamma-carboxyamide group of glutamine residues and primary amines (E. Bendixen et al, J. Biol. Chem. 26821962-21967, 1993; K. N. Lee et al
Biochim. Biophys. Acta
1202 1-6 1993; T. Kanaji et al
J. Biol. Chem.
268 11565-11572 1993). This enzyme could therefore introduce amino acid residues from a peptide into a glutamine residue through a peptide lysine epsilon amino group or into a lysine group via a peptide glutamine group. The enzyme could also catalyse derivatization of glutamine residues with a primary amine.
A further approach is to introduce chemical moieties to either the N or C terminus of a polypeptide using reverse proteolysis or chemical conjugation or a combination of the two (I. Fisch et al,
Bioconj. Chem.
3, 147-153, 1992; H. F. Gaertner et al,
Bioconjug. Chem.
3, 262-268, 1992; H. F. Gaertner et al,
J. Biol. Chem.
269, 7224-7230, 1994; J. Bongers et al,
Biochim. Biophys. Acta,
50, S57-162, 1991; R. Offord,
Protein Engineering,
4, 709-710, 1991). These methods have been used t

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