Bacterial receptor structures

Details Bacterial receptor structures Bacterial receptor structures

1998-05-21

2003-03-18

Minnifield, Nita (Department: 1645)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S300000, C530S825000, C530S402000, C530S387100, C530S386000, C435S007100, C435S173300, C435S069700, C435S882000, C435S883000, C435S320100, C436S536000, C436S501000, C436S518000

Reexamination Certificate

active

06534628

ABSTRACT:

The present invention relates to new bacterial receptor structures originating from natural bacterial receptor structures which have been modified in regard to amino acid residues involved in the original interaction function, whereby said original interaction function has been substantially inhibited and replaced by a modified interaction function directed to a desired interaction partner.
Several bacteria known to invade mammals have evolved surface proteins capable of binding to a variety of substances including host specific carbohydrates and proteins. Several such receptors from Gram-positive bacterial pathogens have been isolated and characterized in detail as will be shown below. Most well-characterized are the Fc receptors, named after the capability of binding to the constant Fc part of IgG. Based on binding experiments to IgG from various mammalian sources, and subclasses thereof, Fc receptors have been divided into six types I-VI. The receptor from
S.aureus,
protein A [SPA], defining the type I receptor, has been the subject of immense studies.
SPA binds IgG from most mammalian species, including man. Of the four subclasses of human IgG, SPA binds to IgG1, and IgG4 but shows very weak or no interaction with IgG3 [Eliasson, M. et al, 1989 J.Biol.Chem. 9:4323-4327]. This pseudoimmune reaction has been used for more than 20 years for the purification and detection of antibodies in diagnostic, research and therapeutic applications. Cloning, sequencing and
Escherichia coli
expression of defined fragments of the SPA gene revealed a highly repetitive organization, with five IgG binding domains [E-D-A-B-C], a cell wall spanning region and membrane anchoring sequence [XM] [Uhlén, et al, 1984 J.Biol.Chem. 259:1695-1702; Moks, T. et al, 1986 Eur.J.Biochem. 156:637-643]. A vast number of plasmid vectors have been constructed, allowing gene fusions to different fragments of the gene for the purpose of fusion protein production in different hosts [Nilsson B. and Abrahmsén, L. 1990 Meth.Enz. 185:144-161] (
FIG. 2
a
).
The structure for a complex between human Fc [IgG1] and a single domain [B] of SPA has been determined by X-ray crystallography at a resolution of 2.8 Å [Deisenhofer, J. et al 1981 Biochemistry 20:2361-2370]. Based on this structure and additional information from NMR experiments, the B domain can be viewed as a compact structure consisting of three anti-parallel &agr;-helices connected with loops. In the Fc binding, which is of both electrostatic and hydrophobic nature, only side chains of residues from helices 1 and 2 are involves, whilst the third helix is not participating in the binding. Based on this domain B, a synthetic IgG-binding domain [Z] [Nilsson, B. et al 1987 Prot,Eng. 1:107-113] has been constructed, suitable as fusion partner for the production of recombinant proteins which allows purification by IgG affinity chromatography. The high solubility and the stable structure of the Z domain has been utilized for production, purification and renaturation of a large number of recombinant proteins. [Josephsson, S. and Bishop, R. Trends Biotechnol. 6:218-224; Samuelsson, E. et al 1991 Bio.Technol. 9:363-366]
Streptococcal strains of serological groups C and G display a binding repertoire for mammalian IgGs, including human IgG3, which is even broader than for the type I receptor. The name protein G was suggested for this type III receptor from group G streptococci. In 1986 Olsson and co-workers reported on the cloning and sequencing of the gene from the serological group G streptococci [G148] [Guss, B. et al, 1987 EMBO J. 5:1567-1575; Olsson, A. et al, 1987 Eur.J.Biochem. 168:319-324]. In analogy with SPA is SPG a repetitively arranged molecule, comprising an IgG-binding region of three homologous domains [C1,C2,C3], spaced by smaller D-regions (FIG.
2
A). Compared to SPA, SPG displays a different binding spectra for immunoglobulins from different species and subclasses thereof. The IgG binding domains of protein G are now widely used as an immunological tool, i.e. in the affinity purification of monoclonal antibodies. Production of subfragments constructed by DNA-technology, have shown that an individual C-region is sufficient for full IgG-binding. Recently, the structure for a complex between the C1-domain from SPG and human Fc was determined with X-ray crystallography (FIG.
2
B). This shows that SPG binds to the CH2-CH3 interface but at a different site compared to SPA. The binding is mainly of electrostatic nature which is in contrast to the large contribution of hydrophobic forces seen for the SPA-Fc interaction. Moreover, the 3-D structure of C1 differs from the X structure in that it is built up by two &bgr;-sheets connected by an &agr;-helix [&bgr;&bgr;-&agr;-&bgr;&bgr;]. The residues of C1 which according to the structure are involved in the binding, corresponds to the &agr;-helix, the loop and the following &bgr;-sheet.
An additional activity of SPG is the capability to bind serum albumin. The binding strength is species dependent, and among the tested samples. SPG binds strongest to serum albumin from rat, man and mouse. Production and binding studies of subfragments of SPG shows that the two binding activities are structurally separated and that the serum albumin binding function is located at the repetitive A-B region [Nygren et al 1990 Eur.J.Biochem. 193:143-148]. This region has been used for several biotechnological purposes. Recombinant proteins have been produced as fusions to the region which enables the purification by affinity chromatography, where human serum albumin most frequently has been used as immobilized ligand. Proteins found to be proteolytically sensitive have been produced as “dual affinity fusions” in which they are flanked by two different affinity tails derived from SPA and SPG, respectively. Purification schemes employing both the N- and C-terminal are thus possible which ensures the recovery of an intact target protein [Hammarberg et al 1989 Proc.Natl.Acad.Sciences USA 86:4367-4371]. The strong and specific binding to serum albumin has also been used for the in vivo stabilization of therapeutic proteins.
Through complex formation with the very long-lived serum albumin, the receptor is carried in the circulation (macque apes) with a half-life which is close to the half-life for serum albumin itself. Studies in mice with the for HIV/AIDS therapy interesting, but rapidly cleared T-cell receptor CD4, showed that it was substantially stabilized when fused to the serum albumin binding region, when compared with an unfused control protein [Nygren et al 1991 Vaccines 91 Cold Spring Harbor Press 363-368]. The slow clearance can probably be explained by the complex formation with serum albumin which circumvents elimination by the liver and excretion in the kidney.
In order to determine the minimal extension required for maintained binding to serum albumin, several smaller fragments of the A-B region have been produced and analyzed. The smallest fragment so far with serum albumin binding activity is a 46 residue fragment [“B2A3”] comprising region A3 flanked by 13 and 9 residues, respectively, from regions B2 and S.
Based on homology and binding studies of other partial fragments SPG is regarded to be trivalent with regard to binding to serum albumin. Similar to the monovalent IgG-binding domains Z and C1 B2A3 is relatively small and shows high solubility and stability and is therefore a suitable candidate for modification.
SUMMARY OF THE INVENTION
The present invention has for its main purpose to provide new bacterial receptor structures by modifying natural bacterial receptors in regard to their original interaction functions to result in new structures having modified interaction functions.
Another object of the invention is to provide artificial bacterial receptor structures which are stable and more resistant to various

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