Commercial production of insulin and insulin-like protein in plants
United States Patent: 7,554,006
Issued: June 30, 2009
Stephen (Bryan, TX), Howard; John (Cayucos, CA)
Assignee: Prodigeue, Inc.
Appl. No.: 12/125,787
Filed: May 22, 2008
Executive MBA in Pharmaceutical Management, U. Colorado
The invention relates to production of
proinsulin or insulin in seeds of monocot plants by transformation of
monocot plants with a nucleotide sequence encoding proinsulin or insulin
under the control of a seed specific promoter.
Description of the
SUMMARY OF THE INVENTION
The invention is the expression of an insulin compound in plants. In a
preferred embodiment, the proinsulin and/or insulin proteins are expressed
in monocotyledonous plants. In a further preferred embodiment, they are
expressed in maize. The proteins may be extracted from the plant, or the
plant tissue used in various applications. In one such application, the
plant tissue can be orally administered to an animal. In yet another
embodiment, a biomass is created by expressing the proteins in a plurality
of plants where at least some of the plants express the proteins, then
harvesting the biomass.
This invention relates to the expression of proinsulin and insulin in
plants, where the plants express at high levels. This invention further
relates to stable transformation of plants with such proteins. As used
herein stable transformation refers to the transfer of a nucleic acid
fragment into a genome of a host organism resulting in genetically stable
The term "insulin" as used herein refers to mammalian insulin, such as
bovine, porcine or human insulin, whose sequences and structures are known
in the art. Bovine, porcine, and human insulin are preferred mammalian
insulins; human insulin is more preferred. The amino acid sequence and
spatial structure of human insulin are well-known. Human insulin is
comprised of a twenty-one amino acid A-chain and a thirty amino acid B-chain
which are cross-linked by disulfide bonds. A properly cross-linked human
insulin contains three disulfide bridges: one between position 7 of the
A-chain and position 7 of the B-chain, a second between position 20 of the
A-chain and position 19 of the B-chain, and a third between positions 6 and
11 of the A-chain. The proinsulin molecule also contains a C-chain, which is
cleaved by enzymes in the human body. Proinsulin is a single polypeptide
chain containing a sequence of about thirty residues that is absent from
mature insulin. Proinsulin has a B-C-A chain structure. The C or connecting
peptide joins the carboxyl end of the B chain and the amino terminus of the
A chain of the future insulin molecule. Biochemistry 3rd edition, pg. 995
(1988) New York, WH Freeman & Co. The mature insulin is generated by
cleavage of the C peptide at dibasic residues after Arg(31)-Arg(32) and
after Lys(64)-Arg(65). Two distinct processing enzymes have been defined
which are specific for their respective dibasic cleavage sites in proinsulin;
type I is substrate specific for the BC junction, while type II is specific
for the CA junction (Weiss, Biochemistry 29, 1990). When proinsulin is used
for manufacture of insulin, enzymes such as trypsin are used to cleave the
C-chain and an enzyme such as carboxypeptidase B is used to further remove
basic amino acids.
Use of insulin analogs is also included within the scope of the invention
and refers to proteins that have an A-chain and a B-chain that have
substantially the same amino acid sequences as the A-chain and B-chain of
human insulin, respectively, but differ from the A-chain and B-chain of
human insulin by having one or more amino acid deletions, one or more amino
acid replacements, and/or one or more amino acid additions that do not
destroy the insulin activity of the insulin analog. Also included in this
invention are analogs of proinsulin, where the C peptide is reduced in
length and/or modified in sequence.
Genes which encode insulin and insulin-like proteins are available to one
skilled in the art. See for example Galloway, J. A. and Chance, R. E., Horm.
Metab. Res. (1994) 26:591-598 and Genbank Access no. XP.sub.--006400;
Arakawa et al., supra; Bell, G. I. et al., (1980) Nature 284: 26-32 (human
insulin gene). Any gene which encodes proinsulin, insulin or insulin analogs
may be used in this invention. Codon optimization for the plant in which the
gene is to be inserted can be useful in obtaining high expression levels, as
can be removing sequences that may destabilize the mRNA.
The methods available for putting together a gene as described above for the
improved expression described above can differ in detail. However, the
methods generally include the designing and synthesis of overlapping,
complementary synthetic oligonucleotides, which are annealed and ligated
together to yield a gene with convenient restriction sites for cloning. PCR-based
approaches may be used to attach and link stretches of sequence. Also,
subsequent PCR amplification of the product may be necessary before later
Once the gene has been made or isolated which encodes the protein, it is
placed into an expression vector by standard methods. The selection of an
appropriate expression vector will depend upon the method of introducing the
expression vector into host cells. A typical expression vector contains
prokaryotic DNA elements coding for a bacterial replication origin and an
antibiotic resistance gene to provide for the growth and selection of the
expression vector in the bacterial host; a cloning site for insertion of an
exogenous DNA sequence, which in this context would code for the protein of
interest; eukaryotic DNA elements that control initiation of transcription
of the exogenous gene, such as a promoter; and DNA elements that control the
processing of transcripts, such as leader and transcription termination/polyadenylation
sequences. It also can contain such sequences as are needed for the eventual
integration of the vector into the plant chromosome.
In a preferred embodiment, the expression vector also contains a gene
encoding a selection marker, which is functionally linked to a promoter and
terminator that control transcriptional initiation and termination,
respectively. For a general description of plant expression vectors and
reporter genes, see Gruber et al. (1993) (Gruber et al., "Vectors for Plant
Transformation" in Methods of Plant Molecular Biology and Biotechnology
89-119, CRC Press, 1993).
Promoter elements employed to control expression of the enzyme encoding gene
and the selection gene, respectively, can be any plant-compatible promoters.
Those can be plant gene promoters, such as, for example, a polyubiquitin
promoter, a promoter for the small subunit of ribulose-1,5-bis-phosphate
carboxylase, or promoters from the tumor-inducing plasmids from
Agrobacterium tumefaciens, such as the nopaline synthase and octopine
synthase promoters, or viral promoters such as the cauliflower mosaic virus
(CaMV) 19S and 35S promoters or the figwort mosaic virus 35S promoter. See
Kay et al. (1987) Science 236:1299 and European patent application No. 0 342
926. See international application WO 91/19806 for a review of illustrative
plant promoters. The range of available plant compatible promoters includes
tissue specific and inducible promoters. In one embodiment of the present
invention, the exogenous DNA is under the transcriptional control of a plant
polyubiquitin promoter. Plant polyubiquitin promoters are well known in the
art, as evidenced by European patent application no. 0 342 926.
Alternatively, a tissue specific promoter can be provided to direct
transcription of the DNA preferentially to the seed. One such promoter is
the globulin promoter. This is the promoter of the maize globulin-1 gene,
described by Belanger, F. C. and Kriz, A. L. (1991) Genetics 129:863-872. It
also can be found as accession number L22344 in the Genebank database.
Another example is the phaseolin promoter. See, Bustos et al. (1989) The
Plant Cell Vol. 1, 839-853.
One option for use of a selectable marker gene is a glufosinate-resistance
encoding DNA and in an embodiment can be the phosphinothricin acetyl
transferase ("PAT") or maize optimized PAT gene (Jayne et al., U.S. Pat. No.
6,096,947) under the control of the CaMV 35S promoter and terminator. The
gene confers resistance to bialaphos. See, Gordon-Kamm et al. (1990) Plant
Cell 2, 603-618; Uchimiya et al., (1993) Bio/Technology 11:835, and Anzai et
al., (1989) Mol. Gen. Genet. 219:492.
It may also be desirable to provide for inclusion of sequences to direct
expression of the protein to a particular part of the cell. A variety of
such sequences are known to those skilled in the art. For example, if it is
preferred that expression be directed to the cell wall, this may be
accomplished by use of a signal sequence and one such sequence is the barley
alpha amylase signal sequence, (Rogers, (1985) J. Biol Chem 260, 3731-3738).
Another example is the brazil nut protein signal sequence when used in
canola or other dicotyledons. Another alternative is to express the protein
in the endoplasmic reticulum of the plant cell. This may be accomplished by
use of a localization sequence, such as KDEL. This sequence contains the
binding site for a receptor in the endoplasmic reticulum. Muntro, S, and
Pelham, H. R. B. (1987) Cell 48:899-907.
Obviously, many variations on the promoters, selectable markers and other
components of the construct are available to one skilled in the art.
In accordance with the present invention, a transgenic plant is produced
that contains a DNA molecule, comprised of elements as described above,
integrated into its genome so that the plant expresses a heterologous
protein-encoding DNA sequence. In order to create such a transgenic plant,
the expression vectors containing the gene can be introduced into
protoplasts, into intact tissues, such as immature embryos and meristems,
into callus cultures, or into isolated cells. Preferably, expression vectors
are introduced into intact tissues. General methods of culturing plant
tissues are provided, for example, by Miki et al., (1993) "Procedures for
Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology
and Biotechnology, Glick et al. (eds) pp. 67-68 (CRC Press 1993) and by
Phillips et al., (1988) "Cell/Tissue Culture and In Vitro Manipulation" in
Corn and Corn Improvement 3d Edit. Sprague et al. (eds) pp. 345-387
(American Soc. Of Agronomy 1988). The selectable marker incorporated in the
DNA molecule allows for selection of transformants.
Methods for introducing expression vectors into plant tissue available to
one skilled in the art are varied and will depend on the plant selected.
Procedures for transforming a wide variety of plant species are well known
and described throughout the literature. See, for example, Miki et al.,
supra; Klein et al., (1992) Bio/Technology 10:286-291; and Weisinger et al.,
(1988) Ann. Rev. Genet. 22: 421-477. For example, the DNA construct may be
introduced into the genomic DNA of the plant cell using techniques such as
microprojectile-mediated delivery, Klein et al., (1992) supra;
electroporation, Fromm et al., (1985) Proc. Natl. Acad. Sci. USA 82:
5824-5828; polyethylene glycol (PEG) precipitation, Paszkowski et al.,
(1984) EMBO J. 3: 2717-2722; direct gene transfer, WO 85/01856 and EP No. 0
275 069; in vitro protoplast transformation, U.S. Pat. No. 4,684,611; and
microinjection of plant cell protoplasts or embryogenic callus, Crossway,
(1985) Mol. Gen. Genet. 202:179-185. Co-cultivation of plant tissue with
Agrobacterium tumefaciens is another option, where the DNA constructs are
placed into a binary vector system, Ishida et al., (1996) Nature
Biotechnology 14, 745-750. The virulence functions of the Agrobacterium
tumefaciens host will direct the insertion of the construct into the plant
cell DNA when the cell is infected by the bacteria. See, for example Horsch
et al., (1984) Science 233: 496-498, and Fraley et al. (1983) Proc. Natl.
Acad. Sci. USA 80: 4803-4807.
Standard methods for transformation of canola are described by Moloney et
al., (1989) Plant Cell Reports 8:238-242. Corn transformation is described
by Fromm et al., (1990) Bio/Technology 8:833-839, and Gordon-Kamm et al.,
The Plant Cell 2:603-618. Agrobacterium is primarily used in dicots, but
certain monocots such as maize can be transformed by Agrobacterium, U.S.
Pat. No. 5,550,318. Rice transformation is described by Hiei et al., (1994)
The Plant Journal 6(2), 271-282, Christou et al., (1991) Trends in
Biotechnology 10:239. Wheat can be transformed by techniques similar to
those used for transforming corn or rice. Sorghum transformation is
described by Wan et al., (1994) Plant Physiol. 104:37. Soybean
transformation is described in a number of publications, including U.S. Pat.
In one preferred method, the Agrobacterium transformation methods of Ishida
supra and also described in U.S. Pat. No. 5,591,616, are generally followed,
with modifications that allow the inventors to recover transformants from
Hill maize. The Ishida method uses the A188 variety of maize that produces
Type I callus in culture. In one preferred embodiment the High II maize line
is used which initiates Type II embryogenic callus in culture. While Ishida
recommends selection on phosphinothricin when using the bar or PAT gene for
selection, another preferred embodiment provides for use of bialaphos
The bacterial strain used in the Ishida protocol is LBA4404 with the 40 kb
super binary plasmid containing three vir loci from the hypervirulent A281
strain. This strain is resistant to tetracycline. The cloning vector
cointegrates with the super binary plasmid. Since the cloning vector has an
E. coli specific replication origin but not an Agrobacterium specific
replication origin, it cannot survive in Agrobacterium without cointegrating
with the super binary plasmid. Since the LBA4404 strain is not highly
virulent, and has limited application without the super binary plasmid, the
inventors have found in yet another embodiment that the EHA101 strain is
preferred. It is a disarmed helper strain derived from the hypervirulent
A281 strain. The cointegrated super binary/cloning vector from the LBA4404
parent is isolated and electroporated into EHA 101, selecting for
spectinomycin resistance. The plasmid is isolated to assure that the EHA101
strain contains the plasmid.
Further, the Ishida protocol as described provides for growing fresh culture
of the Agrobacterium on plates, scraping the bacteria from the plates, and
resuspending in the co-culture medium as stated in the '616 patent for
incubation with the maize embryos. This medium includes 4.3 gl.sup.-1 MS
salts, 0.5 mgl.sup.-1 nicotinic acid, 0.5 mgl.sup.-1 pyridoxine
hydrochloride, 1.0 mgl.sup.-1 thiamine hydrochloride, 1.0 gl.sup.-1 casamino
acids, 1.5 mgl.sup.-1 2,4-Dichlorophenoxyacetic Acid (2,4-D), 68.5 gl.sup.-1
sucrose and 36 gl.sup.-1 glucose, all at a pH of 5.8. In a further preferred
method, the bacteria are grown overnight in a 1 ml culture, then a fresh 10
ml culture is re-inoculated the next day when transformation is to occur.
The bacteria grow into log phase, and are harvested at a density of no more
than OD600=0.6 and preferably between 0.2 and 0.5. The bacteria are then
centrifuged to remove the media and resuspended in the co-culture medium.
Since Hi II is used, medium preferred for Hi II is used. This medium is
described in considerable detail by Armstrong, C. I. and Green C. E.
"Establishment and maintenance of friable, embryogenic maize callus and
involvement of L-proline" Planta (1985) 154:207-214. The resuspension medium
is the same as that described above. All further Hi II media are as
described in Armstrong supra. The result is redifferentiation of the plant
cells and regeneration into a plant. Redifferentiation is sometimes referred
to as dedifferentiation, but the former term more accurately describes the
process where the cell begins with a form and identity, is placed on a
medium in which it loses that identity, and becomes "reprogrammed" to have a
new identity. Thus the scutellum cells become embryogenic callus.
The levels of expression of the gene of interest can be enhanced by the
stable maintenance of a protein encoding gene on a chromosome of the
transgenic plant. Use of linked genes, with herbicide resistance in physical
proximity to the proinsulin or insulin encoding gene, would allow for
maintaining selective pressure on the transgenic plant population and for
those plants where the genes of interest are not lost.
With transgenic plants according to the present invention, protein can be
produced in commercial quantities. Thus, the selection and propagation
techniques described above yield a plurality of transgenic plants, which are
harvested in a conventional manner. The plant with the protein can be used
in the processing, or the protein extracted. Protein extraction from biomass
can be accomplished by known methods which are discussed, for example, by
Heney and Orr, (1981) Anal. Biochem. 114: 92-96.
It is evident to one skilled in the art that there can be loss of material
in any extraction method used. Thus, a minimum level of expression is
required for the process to be economically feasible. For the relatively
small number of transgenic plants that show higher levels of expression, a
genetic map can be generated, via conventional RFLP and PCR analysis, which
identifies the approximate chromosomal location of the integrated DNA
molecule. For exemplary methodologies in this regard, see Glick and Thompson
(1993), in Methods in Plant Molecular Biology and Biotechnology, 269-84 (CRC
One of skill will recognize that after the expression cassette is stably
incorporated in transgenic plants and confirmed to be operable, it can be
introduced into other plants by sexual crossing. Any of a number of standard
breeding techniques can be used, depending upon the species to be crossed.
In one embodiment of the invention, a biomass is created by producing a
plurality of plants by the methods described above, where at least some of
the plants express the proinsulin or insulin proteins. The biomass created
is then harvested. The plants may be used as the source of the proteins,
with all or part of the plant used as the protein source. In a preferred
embodiment of the invention, seed is used as the source of the proteins.
This is particularly preferred when a promoter preferentially expressing the
proteins in the seed is used. Alternatively, the protein may be extracted by
wet milling, dry milling or any one of numerous procedures available.
Introduction of DNA into Plants
Introduction of a nucleotide sequence encoding proinsulin protein in maize
seed was achieved as follows.
The proinsulin-encoding nucleotide sequence used in this example is set
forth in FIG. 1 (see Original Patent) (SEQ ID NO: 1), having been
synthesized for codon optimization in maize, and ensuring no sequences were
present that are predicted may destabilize the mRNA.
The protein encoded by the sequence is set forth in FIG. 2 (see Original Patent)
along with 24 extra N-terminal amino acids encoding the barley alpha amylase
signal sequence (SEQ ID NO: 2). Thus, the proinsulin protein consists of the
86 C-terminal amino acids of the 110 amino acids given in FIG. 2. Once the
signal sequence is lost and proinsulin cleaved, 51 amino acids comprising
insulin will remain. the human form of proinsulin is given.
Immature embryos of corn (Zea mays L.) were isolated from greenhouse-grown
ears at 9-13 days after pollination depending on embryo size, generally
1.5-2.0 mm long. The embryos were treated with A. tumefaciens containing the
DNA construct PGN9079 which carries the proinsulin gene under the control of
maize globulin 1 promoter sequence (PGN9066; Belanger & Kriz, supra). The
construct also contained a barley .alpha.-amylase signal sequence (BAASS;
Rogers et al. 1985 J. Biol. Chem. 260, 3731-3738), for targeting the protein
into the cell wall, and the plant transcription unit (PTU) was terminated by
the pinII terminator, (An et al., 1989, Plant Cell 1:115-122. The PTU was
positioned 5' of a CAMV 35S-pat-35S PTU encoding resistance to the selective
agent bialaphos. See FIG. 3 (see Original Patent).
The treated embryos were plated onto callus induction medium and incubated
in the dark at 19.degree. C. for four days. The embryos were then
transferred to callus maintenance medium (CMM) and cultured in the dark at
28.degree. C. They were transferred every two weeks to fresh CMM medium. The
callused embryos ceased growing after about two weeks on bialaphos and
eventually turned brown. Transgenic calli appeared as early as six weeks
following treatment but the majority of transformation events appeared at
seven to nine weeks after treatment. The transgenic calli were easily
spotted due to their white to pale yellow color, Type II callus phenotype,
and rapid growth rate.
The transgenic events were grown for approximately four more weeks on
bialaphos selection and then plated onto regeneration medium in the dark at
28.degree. C. for somatic embryo production. The somatic embryos were
removed after three weeks and plated onto germination medium in the light
(20-30 .mu.moles sec.sup.-1m.sup.-2) at 25 embryos per plate at 28.degree.
C. The embryos germinated after 7-21 days and the T.sub.0 plantlets were
moved into 25 mm.times.150 mm tubes containing 40 ml of minimal medium and
left in the light as above for at least one week for further shoot and root
The plants were transferred into flats filled with equal parts of SunGro
High Porosity (SunGro Horticulture Inc.) and Metro Mix 700 (Scott's-Sierra
Horticultural Products Co.), covered with humidomes and placed in growth
chambers for three to four weeks at 28.degree. C. and 90 .mu.molessec.sup.-1m.sup.-2.
Humidomes were removed after one week. Plants were transplanted into 2 gal
pots filled with High Porosity potting media and 27 g of Sierra 17-6-12 slow
release fertilizer mixed into the top media surface. Plants were moved to
the greenhouse floor (27.degree. C. and 195 .mu.molessec.sup.-1m.sup.-2).
The T.sub.0 plants were pollinated with pollen from greenhouse-grown maize
plants of elite germplasm.
Claim 1 of 5 Claims
1. A method of producing a protein
comprising a protein selected from the group consisting of proinsulin and
insulin protein, the method comprising providing biomass from a plurality
of monocotyledonous plants comprising plants stably transformed with a
nucleotide sequence encoding said protein, wherein expression of said
protein is directed to seed cells of said transformed plants, and wherein
said protein in at least one of said transformed plants is expressed at
levels of at least 0.20% total soluble seed protein; said protein not
fused to cholera toxin B subunit; and producing said protein in said
transformed plant seed.
If you want to learn more
about this patent, please go directly to the U.S.
Patent and Trademark Office Web site to access the full