Component of stem bromelain

Abstract

A novel protein is a component of stem bromelain and is an anti-cancer agent, an immunostimulant and has antimicrobial activity. The protein may be isolated from stem bromelain by methods such as HPLC.

1 . A protein which is a component of bromelain, has a molecular weight of about 22.2 to 25.08 kDa as determined by SDS-PAGE, has an isoelectric point of about 3.8 to 4.79 and has the amino terminal sequence: 
 Val Pro Gin Ser Ile Asp Trp Arg Asp Tyr Gly Ala Val Asn Glu Cal Lys Asn (SEQ ID NO:1)    and, additionally, contains the following sequences:    Gly Gly Trp Glu Phe Lys (SEQ ID NO: 2)    Lys Ala Val Asn Gly (SEQ ID NO: 3)    Tyr Trp Ile Val Arg (SEQ ID NO: 4)    Asn Ser Trp Gly Ser Ser Trp Gly Glu Gly Gly Tyr Val Arg (SEQ ID NO:5)    Thr Ser Leu Asn His Ala His Thr Ile Ile Val Tyr (SEQ ID NO: 6)    Leu Pro Glu Phe (Gin) Pro (Gin) Val Leu Asp-Ala-(SEQ ID NO: 7)    Gly Val Ser Ser Ser Ser Gly Ala Cys Gly Ile Ala Met Ser Pro Leu-Thr-(SEQ ID NO: 8)    Gly Gly Val Phe Ser Gly Pro Ala Gly (SEQ ID NO: 9)    Asn Asn Ala Tyr (SEQ ID NO: 10)    Ser Ser Gly Thr Lys Tyr Trp-Val-(SEQ ID NO: 11);    where the bracketed amino acids represent alternatives to the preceding amino acid and a “-” represents an unidentified amino acid.    
     
     
         2 . A protein which is a component of bromelain, has a molecular weight of about 22.2 to 25.08 kDa as determined by SDS-PAGE and is obtainable by the following method: 
 i. dissolving bromelain in acetate buffer at pH 5.0;    ii. separating the components of bromelain by fast flow high performance liquid chromatography on S-sepharose eluting with a linear gradient of 0 to 0.8 M sodium chloride in acetate buffer over 300 ml;    iii. collecting the fraction corresponding to the second sharp peak off the column; and    iv. isolating the major protein from the fraction collected in (iii) by anion chromatography and hydrophobic interaction chromatography.    
     
     
         3 . A protein as claimed in  claim 1  or  claim 2  or fruit bromelain protease for use in human or veterinary medicine.  
     
     
         4 . A protein as claimed in  claim 1  or  claim 2  or fruit bromelain protease for use in the treatment or prevention of cancer in a human or other mammal.  
     
     
         5 . The use of a protein as claimed in  claim 1  or  claim 2  or fruit bromelain protease in the preparation of an agent for the treatment or prevention of cancer.  
     
     
         6 . A protein as claimed in  claim 4  or the use as claimed in  claim 5 , wherein the cancer is a solid cancer such as ovarian, colon, breast or lung cancer or melanoma, a non-solid tumour, leukaemia or a virus-induced tumours such as hepatocellular carcinoma, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, Burkitt's lymphoma, Kaposi's sarcoma, T-cell leukaemia or cervical carcinoma.  
     
     
         7 . A protein as claimed in  claim 1  or  claim 2  or fruit bromelain protease for use as an immunostimmulant.  
     
     
         8 . The use of a protein as claimed in  claim 1  or  claim 2  or fruit bromelain protease in the preparation of an immunostimulant.  
     
     
         9 . A protein as claimed in  claim 7  or the use as claimed in  claim 8 , wherein the immunostimmulant is for the treatment of secondary immunodeficiencies arising from malnutrition, infection (for example HIV and malaria), tumours (for example lymphoid, myeloma and other), trauma (for example burns, wounds and surgery), medical treatment (for example with drugs such as steriods, cyclosporin and cyclophosphamide), protein loss (such as in diarrhoea and bums), genetic abnormalities (such as those found in combined immunodeficiency patients who lack T and/or B cells), diabetes and old age.  
     
     
         10 . A protein as claimed in  claim 7  or the use as claimed in  claim 8  wherein the immunostimulant is a vaccine adjuvant.  
     
     
         11 . A protein as claimed in  claim 1  or  claim 2  or fruit bromelain protease for use in the treatment or prevention of diseases or conditions which respond to increased NO production.  
     
     
         12 . The use of a protein as claimed in  claim 1  or  claim 2 , or fruit bromelain protease, in the preparation of an agent for the treatment or prevention of diseases or conditions which respond to increased NO production.  
     
     
         13 . A protein as claimed in  claim 1  or  claim 2  or fruit bromelain protease for use as an anti-microbial agent.  
     
     
         14 . The use of a protein as claimed in  claim 1  or  claim 2 , or fruit bromelain protease, in the preparation of an anti-microbial agent.  
     
     
         15 . Nucleic acid having a sequence which encodes a protein as claimed in  claim 1  or  claim 2  or a sequence complementary thereto.  
     
     
         16 . A pharmaceutical or veterinary composition comprising a protein as claimed in  claim 1  or  claim 2  together with a pharmaceutically or veterinarily acceptable excipient.  
     
     
         17 . A composition as claimed in  claim 16  which is formulated for enteral, for example oral, nasal, buccal, topical or anal administration.  
     
     
         18 . A composition as claimed in  claim 16  which is formulated for parenteral administration, for example by the intravenous, subcutaneous, intramuscular or intraperitoneal routes.  
     
     
         19 . A vaccine composition comprising a protein as claimed in  claim 1  or  claim 2  or fruit bromelain protease as an adjuvant.