Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-11-27
2004-08-03
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S252800, C530S350000, C530S412000
Reexamination Certificate
active
06770457
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process of purifying angiogenesis inhibitor proteins that are expressed in
E. coli
, and also relates to the use of recombinant kringle 1-3 of plasminogen (hereinafter, referred to as “greenstatin”) purified according to the above process as an angiogenesis inhibitor or as an anticancer agent.
BACKGROUND
Angiogenesis is a biological process in which new blood vessel is generated from an existing vessel. Regulated by tight controlling mechanism, angiogenesis is involved in special physiological cases such as wound healing and tissue regeneration. In diseases such as arthritis, diabetic retinopathy, psoriasis, and so on, angiogenesis also happens and converts these diseases into clinical malignancies. Furthermore, angiogenesis is essential to the growth and metastasis of cancer cells. To grow more than 1-2 mm
3
, cancer tissues should be supplied for nutrients, oxygen, and growth actors from new blood vessels induced by angiogenesis (Fidler e, al., Cell, 79 185-88, 1994). Moreover, cancer cells can be metastasized to other tissues through the new blood vessels.
Current cancer therapies mainly include operative therapy, chemotherapy, and radiotherapy. While chemotherapy has the advantage of convenient administration, its disadvantages include nonspecific attack for normal cells, poor uptake on cancer tissue, and drug tolerance. Due to these disadvantages, chemotherapy for cancer is associated with various side effects and poor efficacy, and often cannot be employed repeatedly. Although radiotherapy has been developed, it is also associated with nonspecific effects on normal cells. Thus, anticancer therapy that will cover the side effects of the existing therapies has been required, which will improve current anticancer therapies or make up for their disadvantages.
As a revolutionary anticancer agent, angiogenesis inhibitors have been studied. To treat the angiogenesis-dependent diseases such as cancers, arthritis, diabetic retinopathy, and psoriasis, various angiogenesis inhibitors have been developed last 20 years, and more than 11 inhibitors have been under preclinical or clinical phase. These inhibitors vary in their inhibition mechanisms and inhibition efficiencies.
The angiogenesis inhibitors under clinical or preclinical phase include metastasis inhibitors such as batimastar, cancer growth inhibitors such as interferon and VEGF (; Vascular Endothelial Growth Factor), and natural cryptic proteins such as angiostatin™ (first four kringle domains of plasminogen, SEQ ID NO: 2) and endostatin. Of these inhibitors, angiostatin™ and endostatin are inactive in a native form, but have an angiogenesis inhibitor activity in a cleaved form (Gradishar,
Invest. New Drugs
, 15, 49-59, 1997; Bergers et al.,
Science
, 284, 808-812, 1999).
Angiostatin™ is a protein containing the kringle 1 to 4 region of plasminogen (described by SEQ ID NO: 1), a thrombolysis factor. The amino acid sequence of angiostatin™ (described by SEQ ID NO: 2) is the part (99th~467th amino acid residues) of full plasminogen sequence. On the other hand, greenstatin (described by SEQ ID NO: 3) is a recombinant protein which consists of 254 amino acid residues (101~354). Kringle 4 is deleted in greenstatin but contained in angiostatin™.
The purification of recombinant angiostatin™ is laborious since there are 13 disulfide bonds in the angiostatin™ protein structure. To overcome the difficulties in renaturation of angiostatin™ during purification step, two methods for producing angiostatin™ are currently used. In one method, angiostatin™ is isolated from the plasminogen, as a mixture of kringle 1-3 fragment and kringle 1-4 fragment. The other method exploits yeast or baculovirus system in which recombinant angiostatin™ is produced as a soluble form.
Since greenstatin also has 10 intramolecular disulfide bonds, it is hard to purify and currently expressed and purified in the yeast or baculovirus system. However, on account of the low yield and the high cost, the yeast or baculovirus system is not so efficient and economical.
Although
E. coli
expression system is preferable for the inexpensive and convenient mass-production of angiogenesis inhibitor proteins such as angiostatin™ and greenstatin, it is not suitable for the production of eukaryotic proteins in which sugar chains or the high order of protein folding is required for the activities of the recombinant proteins. Thus, in the
E. coli
system producing the angiogenesis inhibitor proteins, unfolding and refolding processes as well as the expression and purification processes should be optimized to meet with the structural requisite for proper activities.
We, the inventors of the present invention, have investigated the optimized process of purifying the angiogenesis inhibitor proteins such as angiostatin™ and greenstatin, which allows the efficient mass-production of the proteins. In this invention, we construct an
E. coli
expression vector into which DNA sequence encoding an angiogenesis inhibitor protein is inserted; transform
E. coli
with this vector; purify the inhibitor protein from the inclusion bodies of the
E. coli
transformant; perform refolding reaction in the presence of basic amino acids; and perform further purification. This invention is performed by confirming that these processes can be used to massively produce active form of angiogenesis inhibitor proteins, and that the active greenstatin purified according to the processes is applicable for the suppression of angiogenesis in vivo and of angiogenesis-dependent diseases.
SUMMARY OF THE INVENTION
It is an object of this invention to provide the optimized purifying process allowing the efficient mass-production of angiogenesis inhibitor proteins such as angiostatin and greenstatin.
In such aspect of this invention, the process of purifying angiogenesis inhibitor proteins comprises the steps of;
1)constructing an
E. coli
expression vector containing DNA sequence encoding an angiogenesis inhibitor protein, transforming
E. coli
with the vector, and producing the angiogenesis inhibitor protein in the inclusion bodies of
E. coli
transformant;
2)solubilizing the inclusion bodies of step 1);
3)refolding the solubilized fraction of step 2) in a buffer containing urea and glutathione;
It is an additional object of this invention to provide the use of greenstatin purified according to the purifying process for the treatment of cancers including lung cancer, skin cancer, and brain tumor, and of ophthalmic diseases including glaucoma and retinopathy.
Further objects and advantages of the present invention will be disclosed hereinafter.
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Ahn Byung Cheul
Chang Soo-Ik
Hong Yong-Kil
Joe Young Ae
Jung Soo-Il
Caputa Anthony C.
Gates & Cooper LLP
Korea Green Cross Corporation
Rawlings Stephen L.
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