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
1999-09-21
2003-01-07
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C536S023100, C536S024100, C800S280000, C800S298000
Reexamination Certificate
active
06503732
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods for production of recombinant peptides and proteins. More particularly, this invention relates to techniques for inserting peptides into the coat protein of a virus, particularly for purposes of creating a vaccine.
2. Description of Related Art
Tobacco mosaic virus (TMV is a well-characterized plant virus with a single, positive-sense RNA genome of 6395 nucleotides. The sequence of the TMV coat protein was initially derived back in the 1950's, and it was not until 1972 that the structure and roles of the forms of the TMV CP, its assembly and microscopic examination of the polymers were published (Durham, et al.,
J. Mol. Bio.,
67:315-332 and 67:307-314). In 1982 the full genomic sequence of TMV RNA was published, and confirmation of the amino acid sequence of the CP as derived from the sequence of the viral RNA was confirmed (Goelet, et al.,
Proc. Natl. Acad. Sci. USA,
79:5818-5822, 1982). The 126- and 183-kDa proteins, which are required for virus replication (Ishikawa, et al.
Nucleic Acids Res.
14:8291-8305, 1985) are translated directly from the genomic RNA from the same initiation codon. The 30 kDA movement protein (MP), which is involved in cell-to-cell movement, and the 17.5 kDA coat protein (CP) are translated from separate 3′ coterminal subgenomic mRNAs (Siegel, et al.,
Proc. Nat. Acad. Sci. USA,
48:1845-1851, 1976; R. N. Beachy, et al.,
Virology,
63:84-97, 1975.
The life cycle of the TMV virus is well known. The amount of mRNA for the viral proteins determines the amount of each protein produced. The protein produced in the largest amount is the CP, which is as much as 5-10% of the total protein made in the infected cell. The structure of the TMV CP is reviewed in Butler, et al,. (
J. of General Virology,
65:253-279, 1984). The CP encapsidates the viral RNA and is required for long-distance movement in the plant The CP drives the assembly and encapsidation, which in turn enables systemic spread (Dawson, et al.,
Phytopathology,
78:783-789, 1988). Viral assembly and encapsidation are not required for local cell to cell spread of the viral RNA; however, the MP protein, a non-structural protein encoded by the virus, is required for local cell to cell spread of the viral RNA.
A number of research projects have been conducted to establish the use of TMV as a potential vector by inserting peptides into the coat protein. Even before the cloned cDNA of TMV was available, Haynes, et al. (
Bio/Technology,
4:637-641, 1986) attached additional nucleotides at the 3′ end of the coat protein mRNA, resulting in production of a fusion protein carrying sequences to serve as a vaccine for polio virus. The fusion CP was able to assemble and form rod-like structures in
E. coli.
Because the cDNA was unavailable, no work was done in a plant system.
The development of in vitro expression systems that allow production of infectious TMV RNAs from cloned full length cDNA genomes (Dawson, et al.,
Proc. Natl. Acad. Sci. USA,
83:1832-1836, 1986; Meshi, et al.,
Proc. Natl. Acad. Sci. USA,
83:5043-5047, 1986) has permitted the direct manipulation of the TMV genome at the DNA level. Highly infectious RNA transcripts of a full-length infectious cDNA clone of the U1 (common) strain of TMV have been produced in vitro using bacteriophage T7 RNA polymerase (Holt and Beachy,
Virology
181:109-117, 1991) Thus, TMV RNA is a good candidate as a vector for the expression of foreign genes in plants. However, the TMV vectors developed using a CP gene modified to insert foreign genes usually fail to systemically express foreign genes, either through failure to produce intact CP for virus particle formation, or through loss of the foreign gene sequence during replication due to RNA recombination (N. Takamatsu, et al.,
EMBO J.,
6:306-311, 1987; N. Takamatsu, et al,
FEBS Lett.,
2:73-76, 1989; W. O. Dawson, et al.,
Virology,
122:285-292, 1989). One exception is the TMV-ORSV (odontoglossum ringspot virus) hybrid vector, which includes an ORSV CP as an additional intact CP gene, thereby avoiding RNA recombination (J. Donson, et al.,
Proc. Natl. Acad. Sci. USA,
88:7204-7208, 1991). Successful systemic expression was achieved more recently by inserting the “weak” stop codon for the TMV 130 K protein gene, which permits readthrough of the stop codon. immediately after the stop codon for the CP gene and immediately 5′ of a foreign gene sequence encoding a 12 amino acid Angiotensin-I-Converting Enzyme Inhibitor (H. Hamamoto et al.,
Bio/Technology
11:930-932, 1993). Since readthrough occurred relatively rarely, the result was production of a small amount of intact CP as well as the CP fusion protein.
In 1990, a CP fusion protein was described (Takamatsu, et al.,
FEBS Letters,
269:73-76, 1990) containing a five amino acid sequence for enkephalin as a carboxyterminal fusion on the TMV CP. The carboxyl terminus of the CP is known to project from the capsid surface. A methionine sequence placed between the end of the CP and the enkephalin sequence was introduced for isolation of enkephalin in a cyanogen bromide cleavage reaction. During virus infection, a large accumulation in plant cells of virus protein of the expected size was observed However, there was no evidence at this point that the protein could be assembled into virus particles except that one of the coat protein fusion sequences led to systemic symptoms. Other virus showed no systemic mosaic symptoms. Despite these barriers to systemic plant infection with the fusion protein, in infected protoplasts large amounts of the enkephalin fusion protein was produced, and the cyanogen bromide released the protein.
The movement protein of TMV has been studied to determine its function (Deom, et al.,
Cell,
69:221-224, 1992). The mechanism by which the movement protein functions to cause systemic invasion of the virus is unknown. Infection of a plant can be divided into several steps: (i) infection of the first cell; (ii) establishment of a multicellular infection site; (iii) short-distance or cell-to-cell spread through a leaf, which requires the MP; (iv) long-distance spread, which can be subdivided into entry into, travel through, and exit from the vascular system, and which has been shown to involve the CP in
Nicotiana tabacum
(W. O. Dawson, et al.,
Phytopathology
78:783-789, 1988; T. Saito et al.,
Virology
176:329-336, 1990; Takamatsu et al.,
EMBO J.
6:307-311, 1987), and (v) further cell-to-cell spread once the virus has left the vascular system.
The movement protein has a direct effect on the function of plasmodesmata. The molecular size exclusion limit of plasmodesmata in transgenic
Xanthi
tobacco plants expressing the MP gene from the cauliflower mosaic virus (CaMV) 35S promoter has been shown to be at least 10-fold greater than that in control plants (S. Wolf et al.,
Science
24:377-379, 1989). In addition, electron microscopy employing immunogold labeling has shown that the MP is localized in plasmodesmata in tobacco leaf tissue from TMV-infected plants (K. D. Tomenius et al.,
Virology
160:363-371, 1987) and in transgenic plants (D. Atkins et al.,
J. Gen. Virol.
72:207-211, 1991; B. Ding et al.,
Plant Cell
4:915-928, 1992;P. J. Moore et al.,
Protoplasma
170:115-127, 1992). MP produced in and purified from
Escherichia coli
binds single stranded nucleic acids in vitro in a cooperative but non-specific manner and forms a thin extended structure (V. Citovsky et al.,
Plant Cell
4:397-411, 1992).
E. Coli
-produced MP, when injected into a tobacco mesophyll cell, can increase the plasmodesmal size exclusion limit of that cell and of adjacent cells, suggesting that the MP may be able to move from cell to cell in the absence of virus (E. Waigmann et al.,
Proc. Natl. Acad. Sci. USA
91:1433-1437, 1994). The tissues through which TMV must spread in order to successfully infect a plant are not known, In addition, it is not known in which tissues or in what quantities MP must accumulate in order for local or long-
Beachy Roger N.
Fitchen John H.
Davis Katharine F.
Fitting Thomas
Holmes Emily
Schwartzman Robert A.
The Scripps Research Institute
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