Thioredoxin-glutamate decarboxylase 65 fusion protein

Details Thioredoxin-glutamate decarboxylase 65 fusion protein Thioredoxin-glutamate decarboxylase 65 fusion protein

1997-11-28

2004-11-09

Saunders, David (Department: 1644)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007500, C435S007950, C435S069300, C435S069700, C435S002000, C436S506000, C530S350000

Reexamination Certificate

active

06815174

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention refers to a novel hybrid recombinant protein formed by the fusion of the
E. coli
protein thioredoxin (Trx) and the human enzyme, glutamate decarboxylase 65 (GAD65). The chimera Trx-GAD65, which does not exist naturally, is immunologically adequate to replace human GAD65 for the determination of anti-GAD auto antibodies in human serum, with the advantage with respect to natural human GAD65 that it can be produced in
E. coli
cultures with good yield and at low cost. The present invention also includes a method for the production of said protein, alone or in a biotinylated form, as well as applications of said protein in the diagnosis of diabetes mellitus.
Diabetes Mellitus is a severe disease that results in serious consequences for both afflicted individuals as well as society at large. It has been calculated that 5% of the population is afflicted with some type of diabetes. One fifth of the afflicted individuals require, sooner or later, administration of insulin in order to survive. In Argentina, there are more than 300,000 so afflicted individuals. The decision to administer insulin is made by the attending physician as soon as the patient's symptoms indicate the disease is full effect and the patient's pancreatic cells, the insulin producing cells, have been destroyed. At this stage, there is very little that can be done to arrest the progress of the disease.
Fortunately, certain markers have been discovered which permit the early discovery of the probable course of the disease, in individuals predisposed to irreversible forms of the disease, with great anticipation. These genetic and humoral markers allow for the implementation of preventive therapies. Further, animal studies have shown that certain preventative therapies have delayed the onset of the disease for years. Thus far, antibodies against glutamate decarboxylase are among the most efficient markers for insulin dependant Diabetes Mellitus. It would be beneficial to have a specific as well as economic detector for said antibodies.
GAD65 is an enzyme that catalyzes the formation of &ggr;-aminobutyric acid from glutamic acid. It is present in several tissues, including nervous and pancreatic tissues.
Techniques available for the extraction and purification of GAD65 from human tissues do not allow for the extraction of sufficient quantities on the enzyme. Further, such techniques require extraction from human cadavers thereby involving complex legal and technical issues. Natural recombinant enzymes obtained from cell free in vitro systems is adequate for analytical methods, however, said techniques provide low yields and are costly.
Natural recombinant enzyme produced in insect cell cultures result in high yields and are useful for analytical methods, however, the method is lengthy, involved and costly.
Previous attempts at producing, in
E. coli
cultures, natural recombinant GAD65, in either it's native state or in an immunologically competent form for the detection of anti-GAD65, proved unsuccessful. In all those attempts, the resultant GAD65 was irregularly folded, had negligible specific enzymic activity and was recognized by a minority of anti-GAD65 positive patient serums. Other methods involving hybrid systems formed by fusing proteins or peptides to GAD65 were similarly not satisfactory.
Production of recombinant GAD65 in
E. coli
cultures is clearly of benefit from an economic stand point. Accordingly, it would be highly desirable to resolve the problems associated with the production of GAD65 in
E. coli
cultures.
It is known in the art of production of recombinant proteins that the gene of interest may be ligated to a second gene which is expressed satisfactorily in
E. coli
in order to generate a fusion protein and from said fusion protein the desired protein can be obtained. In particular, pTrxFus has been used as an expression vector for the union of genes that code for mammalian growth factors and cytokines (see, “A thioredoxin gene fusion expression system that circumvents inclusion body formation in the
E. coli
cytoplasm”, Biotechnology 11, 187 (1993)). Said publication makes reference to the use of the pTrxFus vector as a means of producing soluble form proteins in an
E. coli
expression system. Further, it also describes the importance of separating the polypeptide from thioredoxin to which it is untied so as result in a practical method for the production of pharmaceutical proteins. Normally, fusion protein is a form of production of the selected protein, that is then separated from the thioredoxin for later use. Separation from the thioredoxin is a necessary step so that it will not interfere with the physico-chemical, biological and immunological properties of the desired protein.
In addition, it is necessary to have an antigen for the detection of anti-GAD antibodies, that is available in quantity, has a high degree of purity, is low cost, and allows one to overcome the difficulties which exist presently in the quantitative assaying of anti-GAD antibodies.
Current techniques for the detection of anti-GAD are radiometric, extremely labor intensive and require highly sophisticated laboratories. Presently, there is no method in the market which is precise, accessible, inexpensive and whose performance characteristics allow for anti-GAD tests on mass scale and away from research laboratories. The present invention has solved one of the principal problems that prevented the development of economic and widely available anti-GAD method of detection: recombinant GAD65 has been produced, in soluble form which is both enzymically and immunologically active, in great quantities and at low cost, as a fusion protein with the thioredoxin peptide.
That the fusion product Trx-GAD65 be expressed correctly in
E. coli
and also conserve, unaltered, it's immunochemical properties was not obvious over the prior art. Particularly in light of prior attempts to express GAD65, alone or as a fusion protein (fused with other peptides other than thioredoxin but used in the art of expression of recombinant proteins).
In addition, the fact that it is not necessary to remove the thioredoxin peptide from the Trx-GAD65 fusion product in order to obtain an adequate antigen could not be anticipated by knowledge of the art.
ELISA assays are easy to perform and highly sensitive. Nevertheless, these type assays in the detection of anti-GAD antibodies present two problems: the first is that an antigen is necessary that is available in sufficient quantity, has a high degree of purity and is of low cost. Therefore, having recombinant protein produced in a prokaryotic system having full immunoreactivity with anti-GAD would satisfy all those requirements and would be ideal for the development and implementation of these assays. Another important problem with ELISA assays is that is many of these assays (particularly in those in which the antigen is adsorbed directly to the solid phase) the antigen loses, either partially or totally, it's native structure. Since serum anti-GAD is directed towards conformational epitopes, loss of native structure could result in loss of immunoreactivity. This would explain why all conventional ELISA for anti-GAD described in the literature to date show a sensitivity (determined in new patients with DMID) of 25 to 30%, which is well below the reference method. In order to resolve this difficulty, at least in theory, the ELISA assay conditions may be altered in order to preserve the structure and access of conformational epitopes of the protein. Among the conditions that may be varied are the ELISA capture methods in which the protein is indirectly joined to the solid phase as well as other alternatives where the antigen and serum are pre-incubated where the antigen-antibody reaction occurs in solution and is then detected via the non-reactive antigen by ELISA.
In, “DELISA: sensitive non-isotopic assay for GAD65 antibodies, a key risk-assessment marker for insulin-dependant diabetes mellitus,” Clinical Chemistry 42:2 263-

Affiliated with

Also associated with

Rating

Thioredoxin-glutamate decarboxylase 65 fusion protein does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Thioredoxin-glutamate decarboxylase 65 fusion protein, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Thioredoxin-glutamate decarboxylase 65 fusion protein will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3362587