DNA encoding f.alpha.-2m-binding protein and protein encoded the

Details DNA encoding f.alpha.-2m-binding protein and protein encoded the DNA encoding f.alpha.-2m-binding protein and protein encoded the

1996-07-03

2000-08-08

Mertz, Prema

Chemistry: molecular biology and microbiology

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

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

435 711, 435 712, 4352523, 43525411, 4353201, 435471, 536 237, 530350, C12N 1511, C12N 1563, C07K 14315

Patent

active

061000558

DESCRIPTION:

BRIEF SUMMARY
The invention relates to the field of gene technology and is concerned with recombinant DNA molecules, which contain nucleotide sequences coding for different proteins or polypeptides which have the ability to specifically bind to .alpha..sub.2 -macroglobulin (.alpha..sub.2 M), a plasma proteinase inhibitor. The invention also comprises microorganisms containing the aforesaid molecules, and the use thereof in the production of the aforesaid proteins or polypeptide.


BACKGROUND OF THE INVENTION

The existence of bacteria that bind specifically to the plasmaproteinase inhibitor .alpha..sub.2 M has earlier been reported (Muller and Blobel 1983; 1985). The binding of .alpha..sub.2 M to streptococci is highly specific and streptococci of different serological groups or species bind the two different conformational forms of this plasma proteinase inhibitor referred to as "slow" (s.alpha..sub.2 M) and "fast" (f.alpha..sub.2 M) based on their electrophoretical behaviour (Muller and Blobel 1983; 1985). The slow form represents the native inhibitor and the fast form the inhibitor/protease complex (Barret et al. 1979). In 1989 Sjobring et al. reported that both conformational forms of .alpha..sub.2 M could be bound to the protein G. This receptor is one of the best studied so called type III Fc receptors (Bjorck and .ANG.kerstrom 1990, Sjobring et al. 1989a, Sjobring el al. 1991). The gene encoding protein G has been cloned and sequenced (Guss et al. 1986; Olsson et al. 1987) and binding properties been studied (Guss et al. 1986; Bjorck et al. 1987; .ANG.kerstrom et al. 1987; Nygren et al. 1988). As a result of these studies it has been shown that protein G binds both IgG and serum albumins through specific domains in the protein. Sjobring et al. 1989b reported that in addition of binding these two serum proteins protein G should also bind to both conformational forms of .alpha..sub.2 M. The latter binding they reported should be located in the IgG binding domain of protein G,--a finding that will be contradicted in the present application. The quantification of free .alpha..sub.2 M and .alpha..sub.2 M-protease complexes using streptococcal .alpha..sub.2 M receptors have been described (Justus et al. 1990). Also in studies concerning purification and characterisation of .alpha..sub.2 M from mastitis milk these streptococcal receptors have been used (Rantamaki and Muller 1992). In these cases intact streptococcal cells have been used as the source of the receptor. Therefore a protein which for instance binds specifically to .alpha..sub.2 M should be of great biotechnological interest. This protein could in analogy to what Nygren et al. 1988 reported be immobilized and used to affinity purify away any undesired .alpha..sub.2 M in the sample. Furthermore different proteins (or fragments thereof) with .alpha..sub.2 M-binding activity could be used to detect and measure the amount of .alpha..sub.2 M in a sample. Generally it may be difficult to obtain a homogeneous and reproducible product if such receptors should be prepared from streptococcal cells directly. Moreover, most streptococci are pathogenic and need complex culture media which involve complications in large-scale cultures. There is thus a need for a new method for producing .alpha..sub.2 M-binding proteins (or fragments thereof).


SUMMARY OF THE INVENTION

The present invention relates to different recombinant DNA molecules comprising nucleotide sequences which code for proteins or polypeptides having .alpha..sub.2 M-binding activity. The natural source of the nucleotide sequences encoding the .alpha..sub.2 M-binding activity is the S. dysgalactiae strain SC1 and/or 8215 respectively, and or the Streptococcus equi subsp. zooepidemicus strain V, but with the knowledge of the respective nucleotide and the deduced amino acid sequences presented here the gene(s) or parts of the gene(s) can be made synthetically or reisolated. In particular the knowledge of the deduced amino acid sequence covering the part of the gene encoding the .alpha..sub.2 M-binding activity

REFERENCES:
patent: 5116964 (1992-05-01), Capon et al.
Barrett et al. The electrophoretically `slow` and `fast` forms of the .alpha..sub.2 -macroglobulin molecule. Biochemical Journal. vol. 181, pp. 401-418, 1979.
Bjorck et al. Streptococcal protein G, expressed by streptococci or by Escherichia coli, has separate binding sites for human albumin and IgG. Molecular Immunology. vol. 24, No. 10, pp. 1113-1122, Oct. 1987.
Gettins et al. Preparation and initial characterization of an intermediate, half-cleaved form of human .alpha..sub.2 -macroglobulin. Biochemistry. vol. 28, pp. 5613-5618, 1989.
Guss et al. Structure of the IgG-binding regions of streptococcal protein G. EMBO Journal. vol. 5, No. 7, pp. 1567-1575, Jul. 1986.
Jonsson et al. MAG, a novel plasma protein receptor from Streptococcus dysgalactiae. Gene. vol. 143, pp. 85-89, May 27, 1994.
Jonsson et al. The type-III Fc receptor from Streptococcus dysgalactiae is also an .alpha..sub.2 -macroglobulin receptor. European Journal of Biochemistry. vol. 220, pp. 819-826, Mar. 15, 1994.
Justus et al. Quantification of free .alpha..sub.2 -macroglobulin and .alpha..sub.2 -macroglobulin-protease complexes by a novel ELISA system based on streptococcal .alpha..sub.2 -macroglobulin receptors. Journal of Immunological Methods. vol. 126, pp. 103-108, 1990.
Sjobring et al. Ig-binding bacterial proteins also bind proteinase inhibitors. The Journal of Immunology. vol. 143, No. 9, pp. 2948-2954, Nov. 1, 1989.
Guss et al, EMBO Journal, vol. 5, No. 7, pp. 1567-1575, Jul. 1986.
Strickland et al., J. Biol. Chem., vol. 265, No. 29, pp. 17401-17404 (1990).
Chatwal et al., J. Bacteriol., vol. 169, No. 8, pp. 3691-3695 (Aug. 1987).
Eaton et al., Cell Biochem. Funct., vol. 7, No. 1, pp. 57-64 (Jan. 1989) (Abstract).

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