Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
1998-12-01
2001-05-15
Borin, Michael (Department: 1631)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S178100, C530S350000, C530S395000, C530S403000
Reexamination Certificate
active
06231862
ABSTRACT:
FIELD OF INVENTION
The present invention relates to a purified new epididymal forward motility protein and a process for the isolation of the said epididymal forward motility protein useful as a fertility promoter/blocker. The process of the present invention particularly relates to a process for the isolation of a purified new epididymal forward motility protein from goat, useful as a fertility promoter as well as a fertility blocker.
BACKGROUND OF THE INVENTION
The forward motility protein (FMP) has potential for use as a contraceptive vaccine to control population growth. FMP has also the potential to rectify some of the problems of human infertility(due to low sperm motility): a great social curse in all human races. It has also the potential for solving some of the problems of animal breeding (due to inadequate sperm motility) thereby enhancing milk and meat production.
Population explosion is a major problem in all developing countries including India. Although several contraceptive methods are available, they have undesirable side effects/lower efficacy. In addition some of these methods are rather expensive.
Another global social problem is human infertility. Approx 15% of couples are believed to be infertile. One reason for human infertility is due to low sperm motility. Although several sophisticated Assisted Reproductive Technologies (e.g. IVF: in vitro Fertilization, ICST: Intracytoplasmic Sperm Injection) are available to solve the problems of oligospermic and asthenozoospermic patients (with low sperm motility), these technologies are highly expensive and the success rate is extremely low.
To solve the problem of population explosion and human infertility, it is essential to have an in-depth understanding of the biochemical basis of the reproductive processes. The following section briefly reviews the literature on epididymal sperm maturation: an important event in the male reproductive function.
Mammalian testicular spermatozoa are immotile and infertile. During transit through epididymis these cells acquire forward motility which is essential for their ability to fertilize the female eggs. Biochemical basis of the epididymal sperm maturation process is not well understood. Reference may be made to Hoskins D. D., Brandt H. and Acott T. S.,
Fed Proc,
37, 2534-2542, 1978; Majumder G. C., Dey C. S., Halder S. and Barua M.,
Arch Androl,
24, 287-303, 1990. Epididymal plasma (EP)/seminal plasma (SP) possess a factor that induces forward motility (FM) in vitro in the caput epididymal immature sperm derived from bull. Reference may be made to Acott, T. S., and Hoskins, D. D.
J Biol Chem,
253, 6744-6750, 1978, Hoskins, D. D., Brandt, H. and Acott, T. S.
Fed. Proc.
37, 2534-2542, 1978. In the case of hamster reference may be made to Cornwall, G. A., Smyth, T. S., Vindivich, D., Harter, C., Robinson, J. and Chang, T. S. K.
Biol Reprod
35, 1065-1074, 1986. And in case of goat reference may be made to Jaiswal, B. S. and Majumder, G. C.,
Int J Androl,
19, 97-102, 1996. Hoskins and his associates (Acott, T. S. and Hoskins, D. D.
J Biol Chem,
253, 6744-6750, 1978, Hoskins, D. D., Brandt, H. and Acott, T. S.
Fed. Proc.
37, 2534-2542, 1978) have partially purified the active principle from bovine SP and EP and it has been designated as forward motility protein (FMP). FMP originates from epididymis, after its synthesis in epididymis the factor is secreted into epididymal plasma and subsequently it passes to seminal plasma (Brandt, H., Acott, T. S, Johnson, D. J. and Hoskins, D. D.
Biol. Reprod.
19, 830-835, 1978). For the isolation of FMP, these investigators have used several fractionation techniques as elaborated below. The bovine SP was heated at 90° C. for 10 min. and then centrifuged at 1,78,000 xg for 60 min to remove the precipitated proteins. The resulting supernatant fluid was subjected to Sepharose 6B chromatography using a buffer containing 4M urea, 100 mM NaCl, 10 mM dithiothreitol and 100 mM Tris-HCl, pH 7.5. By these procedures FMP was purified to about 15-fold. Alternatively the factor was also partially purified further by concanavalin A-agarose affinity chromatography of the heat-treated SP. FMP binds to concanavalin A and was eluted with 0.25 M-&agr;-methyl -D-mannoside. The isolated FMP contained several proteins as shown by sodium dodecyl sulphate (SDS)-gel electrophoresis and the extent of purity of FMP is not known. By using similar methods FMP was isolated from bovine EP although the degree of its purification is not known. FMP is a 37 KDa heat-stable glycoprotein that is believed to be essential for the initiation of sperm FM during the epididymal sperm maturation. FMP induces forward motility in vitro in the bovine immature/immotile caput-epididymal spermtozoa.
OBJECT OF THE INVENTION
The main object of the present invention is to provide a process for the isolation of a purified new epididymal forward motility protein (FMP) from goat, specifically derived from EP of goat epididymis. Another object is to provide a process for the isolation of a novel FMP (125 KDa) from goat EP which is strikingly different from bovine FMP, as mentioned above. By the process of the present invention for the first time we have achieved complete purification of FMP: the physiological sperm motility initiator/activator from EP. The isolated FMP obtained by the process of the present invention is homogeneous as evidenced by native polyacrylamide gel electrophoresis (PAGE), high performance liquid chromatography (HPLC) and iso-electricfocusing techniques, implicating thereby that the purity of FMP is nearly 100%. The FMP (125 KDa) obtained is different from the 37 KDa FMP partially purified from bovine SP by Acott T. S. and Hoskins D. D.,
J Biol Chem,
253, 6744-6750, 1978. FMP is highly immunogenic and its antibody strongly inhibits FM of mature sperm thereby implicating that FMP has the potential to serve as a contraceptive vaccine. As the protein obtained by the process of the present invention has high efficacy for stimulating motility of mature spermatozoa, FMP has potentially for rectifying some of the problems of human infertility due to low sperm motility. The goat epididymal FMP obtained by the process of the present invention has a strikingly different molecular mass as compared to the serum motility-promoting proteins of human (approx 200 KDa). Reference may be made to Pat. No. WO 9012032, Oct. 18, 1990, U.S. Pat. No. 5,304,464, Apr. 19, 1994; U.S. Pat. No. 5,453,354, Sep. 26, 1995. It is also different from that from buffalo serum (66 KDa) which has been described and claimed in our Patent Application No. 637/DEL/96 dated Mar. 27, 1996 that has the potentiality for the treatment of human infertility due to low order of sperm motility.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a purified new epididymal forward motility protein and a process for the isolation of the said epididymal forward motility protein useful as a fertility promoter/blocker which comprises subjecting goat epididymal plasma to multiple fractionations till purification to obtain purified new epididymal forward motility protein.
In an embodiment of the present invention the fractionation may be effected using methods such as ammonium sulphate fractionation, cation-exchange chromatography using diethylaminoethyl (DEAE)-cellulose/Sephadex as the ion-exchange resin, anion-exchange chromatography using carboxymethyl (CM)-cellulose/Sephadex as the ion-exchange resin, concanavalin A-Sepharose 4B affinity chromatography, chromatofocusing using polybuffer exchanger termed as PBE-94 resin, adsorption chromatography using alumina gel or hydroxyapatite of BioRad and molecular sieving chromatography using Sephacryl/Sephadex as the insoluble matrix of high performance liquid chromatography. The process of this invention comprises of (a) purification and characterization of a novel FMP and (b) the methodologies for its purification from goat EP. Our invention reports for the first time the purification to apparent homogeneity of FMP from EP. The purity of the isolated
Jaiswal Bijay Shankar
Majumder Gopal Chandra
Borin Michael
Council of Scientific and Industrial Research
Ladas & Parry
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