Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
2001-05-17
2002-12-17
Guzo, David (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093100, C424S093600, C435S455000, C435S456000, C435S325000, C435S320100, C435S069100, C435S348000, C536S023100, C536S023500, C536S024100
Reexamination Certificate
active
06495132
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. §119 of Japanese application number P200-144518 filed May 17, 2000, the contents of which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates to a method for producing polypeptides. More particularly, the present invention relates to the method for producing polypeptides in insects and/or cultured insect cells by recombination technology to enhance the efficiency of production of the polypeptides.
BACKGROUND OF THE INVENTION
Many methods for producing a polypeptide or a protein have been developed up to now. For example, taking a protein, a biological component as an example, it is possible to prepare the protein sample directly from living body, but it has generally some problems in large scale production, high cost as well as heterogeneity due to the presence of isoforms in the case of enzymes. Recently the genetic engineering method is most frequently used. In that case, the method using
E. coli
is mostly employed.
E. coli
is easy to handle and the expression in the
E. coli
-based system is most rapid, but it has also some problems. Because
E. coli
is a prokaryote, there frequently occurred the problems such that water insoluble protein is formed due to unsuitable folding when the protein is an exogenous protein from a eukaryote and that modifications of the protein that are important for its function do not take place after translation in
E. coli
cells. In that case, the method using baculovirus to express such a protein in a cultured insect cell is usually employed for avoiding the above problems. However, in this system, small amounts of the expressed protein provide a perplexing problem.
SUMMARY OF THE INVENTION
Accordingly, the object of the present invention is to provide a method to solve the above problems that amounts of the expressed protein are small when the protein is expressed in an insect or a cultured insect cell by using baculovirus.
The present inventors carried out the expression of tropomyosin protein from several kinds of organisms and surprisingly found that only the amount of tropomyosin from lobster is remarkable and more than 30-fold high as compared with, for example, that from rabbit. Based on that finding, the present inventors studied more and more and found the untranslated polynucleotide sequence from lobster has a function enhancing the expression of proteins. The present invention is based on these findings and accomplished by further studies.
Accordingly in the first aspect of the present invention, there is provided a base sequence of SEQ ID NO:1 which is an untranslated leader sequence from lobster having a function enhancing the expression of a coding sequence located downstream thereof. All the base sequences obtained by deletion, substitution, insertion and/or addition of one or more nucleotides in the base sequence set force in SEQ ID NO:1 are also included in the present invention as far as these base sequences have a function enhancing the expression of a coding sequence located downstream thereof.
In the second aspect of the present invention, there is provided a transfer vector obtained by inserting the base sequence of SEQ ID NO:1 or the base sequence obtained by deletion, substitution, insertion and/or addition of one or more nucleotides in the base sequence set force in SEQ ID NO:1 having a function enhancing the expression of a coding sequence located downstream thereof and a DNA sequence coding a polypeptide of interest ligated downstream of said base sequence into the position downstream of the promoter sequence from baculovirus. Such promoter sequences from baculovirus include polyhedrin gene sequence. This promoter is used for integration of the transfer vector into baculovirus genome. Such vectors containing the promoter from baculovirus include, for example pVL1392 vector. When this vector is used, it is preferable to insert the sequence of SEQ ID NO:1 and the like of the present invention and a DNA sequence coding a polypeptide of interest ligated downstream thereof in the multicloning site of the vector.
In the third aspect of the present invention, there is provided a recombinant baculovirus genome capable of expressing a polypeptide of interest, which is constructed by integrating the base sequence relating to the expression of the polypeptide of interest from the transfer vector of the present invention into a baculovirus genome by in vivo homologous recombination. The homologous recombination may occur in an insect or a cultured insect cell or in a yeast cell or an
E. coli
cell. The insect mentioned above is preferably silkworm and the cultured insect cell is preferably a Sf9 cell, Sf21 cell or High five (trademark) cell (Invitrogen). The base sequence relating to the expression of polypeptide of interest may be inserted into baculovirus genome by means of trans element from transposon in
E. coli
cells. In this case, a commercially available kit may be used.
In the fourth aspect of the present invention, there is provided a recombinant baculovirus having the recombinant baculovirus genome of the present invention. The recombinant baculovirus can be obtained by transfecting the recombinant baculovirus genome prepared in the third aspect of the present invention into an insect or a cultured insect cell capable of being host for baculovirus.
In the fifth aspect of the present invention, there is provided a method for producing a polypeptide of interest comprising a step of infecting an insect or cultured insect cell with the recombinant baculovirus of the present invention to express the polypeptide of interest in the cell and a step of collecting the expressed polypeptide.
The polypeptide of interest in the above second to fifth aspects of the present invention includes interferon &bgr;, interferon &ggr;, interleukin-1 &agr;, interleukin-1 &bgr;, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, cow-somatotropin, erythropoietin, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor XIII, skeletal growth factor, colony-stimulating factor, epithelial growth factor, growth hormone releasing factor, fibroblast growth factor, tissue plasminogen activator, transforming growth factor, thrombopoietin, luciferase and the like as well as tropomyosin, but is not limited thereto. Among them, glycoprotein such as interferon &bgr;, interferon &ggr;, interleukin-1 &agr;, interleukin-1 &bgr;, interleukin-5, erythropoietin, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, blood clotting factor VII, blood clotting factor VIII, blood clotting factor IX, blood clotting factor XIII, tissue plasminogen activator, thrombopoietin and the like are preferable as the protein of interest for the present invention.
All patent applications, patents and literature references cited herein are hereby incorporated by reference in their entirety.
REFERENCES:
Donald L. Mykles et al, Cloning of tropomyosins from lobster (Homarus americanus) striated muscles: fast and slow isoforms may be generated from the same transcript, Journal of Muscle Research and Cell Motility 19, 105-115, (1998).
Maeda Kayo
Maeda Yuichiro
Sano Ken-ichi
Guzo David
Heslin Rothenberg Farley & & Mesiti P.C.
Riken
LandOfFree
Method for producing polypeptides does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for producing polypeptides, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for producing polypeptides will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2998101