Expression of somatotropin in plant seeds

Details Expression of somatotropin in plant seeds Expression of somatotropin in plant seeds

1998-12-18

2001-09-11

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

Nonplant protein is expressed from the polynucleotide

C800S278000, C800S287000, C800S306000, C800S310000, C800S312000, C800S313000, C800S314000, C800S320100, C800S322000, C435S069400, C435S069700, C435S468000, C536S023400, C536S023510, C536S023600

Reexamination Certificate

active

06288304

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides novel transgenic plant seeds comprising a somatotropin as well as methods of preparing plant seeds comprising somatotropins.
BACKGROUND OF THE INVENTION
Naturally occurring somatotropins are polypeptides, the amino acid sequences of which for a number of vertebrate species have been reported. These include bovine (Miller et al., 1980, J. Biol. Chem, 255, 7251) porcine (Seeburg et al., 1983, DNA 2: 37), human (U.S. Pat. No. 3,853,832; Martial et al., Science, 205: 602-617) and various piscine somatotropins (e.g.: Sekine et al., 1985. Proc. Natl. Acad. Sci. (USA), 82: 4306-4310; Agellon et al., 1988, Proc. Natl. Acad. Sci. (USA), 85: 5136-5140; U.S. Pat. Nos. 4,689,402 and 4,894,362). In general, somatotropins isolated from different species display a high degree of amino acid sequence identity (Chang et al., 1992, Gen. and Comp. Endocrin. 87: 385-393). Analogs of somatotropins are also known. European Patent Application 103 395, for example discloses bovine somatotropin analogs. These analogs typically relate to the insertion, addition or deletion of nucleotides of the somatotropin gene thereby creating a protein different from the naturally occurring somatotropin.
The preparation of somatotropins is well known in the art. Bovine somatotropin, for example can be prepared by extraction from the pituitary tissue, (Li et al., 1954, J. Biol. Chem. 211: 55 and U.S. Pat. No. 4,371,462). Somatotropins can also be prepared by production in genetically engineered microorganisms, such as
Escherichia coli
containing recombinant DNA which encodes a somatotropin polypeptide (e.g. Seeburg et al., 1978, Nature). U.S. Pat. No. 4,443,549 discloses a method for producing bovine somatotropin in yeast cells. Methods for high yield bovine somatotropin production in microorganisms are disclosed in U.S. Pat. Nos. 5,240,837 and 5,489,529.
Similarly, the preparation of human somatotropin is known. For example U.S. Pat. No. 5,637,495 and Mukhija et al. (Gene 165: 303-306) teach the production of human growth hormone in
E. coli.
Fish somatotropins have also been produced in a variety of microorganisms. U.S. Pat. No. 5,270,180 for example discloses a method for the production of salmon growth hormone in
E. coli
and yeast and in U.S. Pat. No. 4,894,362 a microbial production system for eel growth hormone is disclosed.
The low costs associated with growing plants, make plants an attractive host for the production of somatotropins. To the best of the present inventors knowledge only one attempt has been reported to produce a somatotropin in plants. Bosch et al. (Transgenic Research, 1994, 3: 304-310) expressed a trout growth hormone in the leaves of transgenic tobacco plants, however they were unsuccessful in accumulating somatotropin in seeds.
Although methods for producing somatotropins are well known to skilled artisans, the existing methods are relatively expensive, especially when large production volumes are required. Accordingly there is a need in the art for additional economical production methods of somatotropin.
SUMMARY OF THE INVENTION
The present inventors have discovered a cost effective method for the preparation of somatotropins in the seeds of plants. The method involves expressing a somatotropin in plant seeds as an oleosin fusion protein so that the somatotropin has biological activity.
Accordingly, the present invention provides a method for the expression of a somatotropin in a plant comprising:
(a) introducing into a plant cell a chimeric nucleic acid sequence comprising:
(1) a first nucleic acid sequence capable of regulating the transcription in said host cell of
(2) a second nucleic acid sequence, wherein said second sequence encodes a recombinant fusion polypeptide and comprises (i) a nucleic acid sequence encoding a sufficient portion of an oleosin protein to provide targeting of the recombinant fusion polypeptide to a lipid phase, linked in frame to (ii) a nucleic acid sequence encoding said somatotropin; and
(3) a third nucleic acid sequence encoding a termination region functional in said plant cell; and
(b) growing said plant cell to produce said recombinant fusion polypeptide.
In a preferred embodiment of the invention, the somatotropin is fish growth hormone. In a further preferred embodiment of the invention the somatotropin is carp growth hormone.
In another aspect the invention provides a chimeric nucleic acid sequence, capable of being expressed in association with an oil body of a plant cell, comprising:
(1) a first nucleic acid sequence capable of regulating the transcription in said plant cell
(2) a second nucleic acid sequence, wherein said second sequence encodes a recombinant fusion polypeptide and comprises (i) a nucleic acid sequence encoding a sufficient portion of an oleosin protein to provide targeting of the recombinant fusion polypeptide to a lipid phase, linked in reading frame to (ii) a nucleic acid sequence encoding a somatotropin; and
(3) a third nucleic acid sequence encoding a termination region functional in said host cell.
In a further aspect, the instant invention provides plant seeds expressing a somatotropin. In one embodiment of the invention the somatotropin is a fish growth hormone.


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