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
1999-09-27
2002-11-12
Benzion, Gary (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S041000
Reexamination Certificate
active
06479257
ABSTRACT:
DETAILED DESCRIPTION OF THE INVENTION
1. Technical Field to which the Invention Belongs
The present invention relates to a method for converting protein produced in a biologically inactive form into natural-form protein (native-form protein) having activity when the protein is in a biologically inactive non-natural state (non-native-form) for the reason that its stereostructure is a higher-order structure biologically different from that of the natural protein having a normal biological activity, or for any other reasons.
The present invention relates to a method for converting protein which for some reasons, is rendered biologically inactive thus failing to have a biological activity which it would have in its normal stereostructure, into biologically active protein, as well as to an apparatus therefor. More specifically, the present invention relates to a method for activating protein, e.g., produced in transformant by means of genetic engineering and rendered biologically inactive due to formation of a stereostructure different from that of its natural protein.
2. Prior Art
Enzymes derived from organisms, peptides and proteins having a biological activity such as cytokine, came to be producible in a large amount by use of microorganisms, animal and plant cultured cells. However, it is known that heterologous proteins expressed in microorganisms as hosts often undergo denaturation in the host cells and occur in biologically inactive forms as precipitates. Further, even if produced and secreted extracellularly, the presence of peptides or proteins in biologically inactive forms are also known. One speculative reason is that although the proteins have the same primary structures as those of their natural types, their stereostructures are biologically not correctly constituted, thus failing to attain structures having biological activities.
Further, it is known that chemically synthesized polypeptides and biosynthesized proteins in cell extracts (cell-free system) often fail to have correct stereostructures, and these have, e.g., inactive structures with relatively less folding lacking in disulfide linkages.
JP-A 59-161321 discloses a method for restoring the activity of protein in a biologically inactive state failing to attain a correct stereostructure owing to incomplete disulfide linkages, wherein precipitated inactive protein is solubilized with a strong detergent, then the solubilized fluid is diluted or the strong detergent is replaced by a weak detergent thereby making the inactive protein active, and the biologically active protein is then recovered. However, this prior art method suffers from the problems that the volume of the protein solution is significantly increased and the activation is time-consuming. Further, because it is difficult to derive a general principle applicable in common to activation of a wide variety of proteins in said method, much endeavor as well as repeated trial and error are actually required for determining conditions for each individual protein of interest. No satisfactory method has been developed.
PROBLEMS TO BE SOLVED BY THE INVENTION
To raise the yield of a target protein produced by, e.g., recombinant DNA technology, it is conventional to take approaches to improving a gene, e.g., for modification of a signal peptide or to improving a method of culturing a transformant. Contrary to the prior art, the present application is drawn to an indirect method for raising the yield of target protein not by directly raising the yield thereof, but by converting inactive protein produced in a culture into an active type, and the object of the present invention is to provide a method for activating protein not having a biological activity for reason of failing to have an active stereostructure or for any other reasons, so that the protein can be converted into biologically active protein very easily and rapidly as compared with the previously proposed or practiced methods.
Further, the present invention provides a method for separating and fractionating protein produced in a biologically inactive form from a solution containing both protein produced in a biologically active form and the protein produced in a biologically inactive form, wherein the protein produced in a biologically inactive form resulting from, e.g., incorrect disulfide linkages is specifically aggregated, precipitated and recovered. As a matter of course, the inactive protein separated and fractionated in this manner can also be activated by the activation method described above.
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Miyauchi Akira
Mizukami Makoto
Ozawa Makoto
Yoshida Masato
Benzion Gary
Higeta Shoyu Co. Ltd.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Strzelecka Teresa
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