Anti-bacterial protein extracts from seeds of marigold and paprika

United States Patent 6086885

The present invention describes the method of extraction and the effect of a
crude protein extract from the seed tissue of two botanical species, Tagetes and
apsicum, on the survival of two economicall! important gram"negative
bacteria Salmonella t!phimurium and #scherichia coli$
US Patent References:
%ethod for preparing a cell free homogenate of hr!santhemum
cinerariaefolium &Trev$' (occ$ containing en)!mes and methods of use
*ito et al$ " +une, ,-85 " .5/5.55
0emicellulase supplement to improve the energ! efficienc! of hemicellulose"
containing animal feed
1odge et al$ " +ul!, ,--5 " 5./-8/8
(iocidal proteins from plants
(roe2aert et al$ " %a!, ,--6 " 55,.33-
(iocidal proteins
(roe2ert et al$ " +ul!, ,--6 " 55485/5
Pesticide product derived from the plant Tagetes minuta
52ioga et al$ " September, ,--3 " 566/-,5
6nventors7
*iegenfuss, Steve &8es %oines, 69'
(rin2haus, 1riedhelm &Urbandale, 69'
:reaves, +ohn &9n2en!, 69'
9pplication ;umber7
0-<053854
Publication 8ate7
03<,,</000
1iling 8ate7
0.<0-<,--8
=iew Patent 6mages7
8ownload P81 6086885 P81 help
#xport itation7
lic2 for automatic bibliograph! generation
9ssignee7
>emin 6ndustries, 6nc$ &8es %oines, 69'
Primar! lass7
./.<360
5ther lasses7
./.<36., 5,.</, 540<430
6nternational lasses7
A61K36/81? 90,;65<00? 96,>45<38
1ield of Search7
./.<,-5$,, 5,.</, 540<430
5ther @eferences7
aceres et al$ APlants used in :uatemala for the treatment of gastrointestinal
disorders$ ,$ Screening of 8. plants against enterobacteriaA$ +ournal of
#thnopharmacolog!, ,--0, vol$ 40, ;o$ ,, pp$ 55"34$
=icente et al$ A#ffect of administration of dietar! papri2a seed on Salmonella
enteriditis on broiler chic2sA, ,--5$ Proceeding of the 1ort!"1ourth Bestern
Poultr! 8isease conference, %ar$ 5"3, ,--5$ ;o$ ..th, p$ ,/-$
Pellicer et al$ Aapsaicin or 1eeding with @ed Pepers 8uring :estation
hanges the Thermonociceptive @esponce of @at 5ffspringA, Ph!siolog! and
(ehavior, 9ug$ ,--6, vol$ 60, ;o$ /, pp$ .45".48$
Primar! #xaminer7
Saucier, Sandra #$
9ssistant #xaminer7
9fremova, =era
9ttorne!, 9gent or 1irm7
0erin2, #sC$ >ent 9$
Parent ase 8ata7
This application claims the benefit of Provisional 9ppl$ Ser$ ;o$ 60<0.4,//5,
filed 9pr$ ,0, ,--3$
laims7
Be claim7
,$ 9 proteinaceous extract which has antibacterial activit! against gram"
negative bacteria made b! the process of7
extracting seeds from Tagetes sp$ or apsicum sp with an aCueous solution to
form a first supernate and insolubles,
separating the first supernate from the insolubles?
adding ammonium sulfate to 40D relative saturation as a precipitating agent
thereb! precipitating a first fraction and forming a second supernate?
collecting the second supernate?
adding amonium sulfate to 30D relative saturation as a precipitating agent
thereb! precipitating a second fraction and forming a third supernate?
dissolving the second precipitate fraction in an aCueous medium?
heating the aCueous medium, which contains the second precipitate fraction, to
80E $ thus forming a third precipitate and a third supernate?
separating the third supernate?
dial!)ing the third supernate using a ,000 8alton molecular weight cut off
membrane? and
collecting the dial!)ed supernate which does not pass through the membrane$
/$ The proteinaceous extract of claim , wherein the gram"negative bacteria are
selected from the group consisting of #scherichia coli, Fisteria and
Salmonella$
4$ The proteinaceous extract of claim ,, wherein the gram"negative bacteria is
strain 0,53703 of #scherichia coli$
.$ The proteinaceous extract of claim ,, wherein the seeds are from Tagetes sp$
and the gram"negative bacteria is Salmonella t!phimurium$
5$ The proteinaccous extract of claim ,, wherein the gram"negative bacteria is
Fisteria monoc!togenes$
6$ The proteinaccous extract of claim ,, wherein the Tagetes sp$ is Tagetes
erecta$
3$ The proteinaceous extract of claim ,, wherein the apsicum sp is apsicum
annum$
8$ 9n animal feed composition having anti"bacterial activit! comprising a feed
to which the proteinaccous extract of claim , is added in an amount effective to
reduce populations of gram"negative bacteria in an animal$
-$ 9 method of reducing a population of gram negative bacteria in an animal
comprising adding the proteinaceous extract of claim , to a feed and orall!
administering the feed to the animal in an amount effective to reduce the
population of gram"negative bacteria in the animal$
,0$ The method of claim -, wherein the proteinaceous extract, after free)e
dr!ing, is added in an amount of between 0$0, ppm and ,000 ppm b! weight of
the untreated feed$
,,$ 9 method of reducing the bacterial load of meat comprising adding the
proteinaceous extract of claim , to a feed and orall! administering the feed to
an animal to be used for meat in an amount effective to reduce the population
of gram"negative bacteria in the animal$
8escription7
16#F8 51 T0# 6;=#;T65;
The present invention relates to a botanical extract of protein from marigold or
papri2a that has anti"bacterial activit!$ %ore specificall!, the present invention
relates to proteins having anti"bacterial activit! against the gram"negative
bacteria Salmonella sp$ and #scherichia coli$ The present invention describes
the method of extraction and the effect of a protein extract from the seed tissue
of Tagetes sp$ or apsicum sp$ on the survival of two economicall! important
bacteria Salmonella t!phimurium and #$ coli strain 0,5303$ The present
invention includes a method for reducing bacteria in animals and animal
products, a method of extraction of the genes encoding for the subGect proteins,
and a method of use of such genes transformed and expressed in grain products
such that the resultant grain has anti"bacterial activit!$
SU%%9@H 51 T0# 6;=#;T65;
The present invention describes the method of extraction and the effect of a
crude protein extract from the seed tissue of two botanical species on the
survival of two economicall! important gram"negative bacteria Salmonella
t!phimurium and #scherichia coli$ (oth bacterial organisms are extremel!
common in animal feed and production animals and are of grave concern in
animal agriculture$ Transmitted through the food chain, both bacterial
organisms cause food poisoning in humans, and control of these organisms is a
public health issue of high priorit!$ The! also cause economic losses in
production animal agriculture through pathogenic effects on the gastrointestinal
tracts of monogastric animals, such as poultr! and swine$ The anti"bacterial
proteins are derived from the seeds of marigold &Tagetes sp$' or papri2a
&apsicum sp$'$
There are several s!nthetic chemicals such as organic acids and formaldeh!de
which can be used to control Salmonella and #$ coli in feeds$ 0owever, these
chemicals are not without their own concerns on the health of animals and
humans$ 9lso, secondar! plant products contained in an essential oil fraction
have been isolated from man! botanical species, among them Tagetes sp$, and
are 2nown to exhibit strong antimicrobial as well as antifungal activities$
(roe2aert et al$, U$S$ Pat$ ;os$ 5,548,5/5 and ,5,.,33- &,--6' demonstrated
the use of a range of specific peptides derived from botanical species belonging
to the (rassicaceae, ompositae and Feguminosae families including
@aphanus, (rassica, Sinapis, 9rabidopsis, 8ahlia, nicus, Fath!rus, litoria,
9maranthus, apsicum, (ri)a and related species on several fungal organisms
and on gram"positive bacteria$ 0owever, the! did not describe an! effect on
gram"negative bacteria$ To our 2nowledge there are no documented examples
of natural, plant"derived proteins or peptides which have a significant biocidal
effect on gram"negative bacteria such that the! could be used to control these
organisms in a range of applications$ 6n addition, there are no reports on
antimicrobial or antifungal proteins characteri)ed in Tagetes$ The present
invention describes novel proteins from two botanical sources and their effect
on more important gram"negative bacteria$
The proteins extracted from the seeds of such botanical species can be used to
control Salmonella and #$ coli in animal feeds and human foods$ 1urthermore,
the proteins and the genes that code for them could be seCuenced and
manipulated via genetic engineering techniCues for expression in production
microorganisms via fermentation$ 6n addition, the genes could be expressed in
the seeds of economicall! important crops such as corn, so!bean, wheat and
rice for inclusion of such anti"bacterial proteins in the downstream processing
and use of these grains in animal feed and human food$ Production of proteins
via genetic engineering of microbes and plants followed b! fermentation or
agronomic production is now common place and established practice$
5verproduction of secondar! plant products via pathwa! engineering, on the
other hand, is still technicall! a challenge$
(@6#1 8#S@6PT65; 51 T0# 8@9B6;:S
16:$ , is a graphical representation of anti"bacterial inhibition data of the
proteins of the present invention$
8#T96F#8 8#S@6PT65; 51 T0# 6;=#;T65;
The present invention relates to a crude protein extract, the purified protein&s',
and the gene&s' encoding for the protein that are extracted from marigold and
papri2a$ The protein inhibits the activit! of #$ coli &strain 0,5303' and possibl!
also S$ enteritidis and S$ choleraesuis$ The protein also inhibits Salmonella
t!phimurium and Fisteria monoc!togenes$
The present invention is protein&s', and the gene encoding such proteins, which
inhibit the growth of the gram"negative bacteria$ These proteins are naturall!"
occurring in the seeds of marigold$ These proteins can be used in the raw form,
as a crude protein extract, and applied to animal feed or human foodstuff
&particularl! those foodstuff that are high in #$ coli or Salmonella
t!phimurium$'$ The proteins can be purified and applied to feed for animals and
foodstuff for humans$ 9lternativel!, the proteins can be applied directl! to
places in which bacteria multipl!$
The genes encoding for these proteins can be transformed into bacteria, fungi,
!easts, plants, or mammals &pro2ar!otic or eu2ar!otic cells', and the protein
can be harvested and applied directl! to the bacteria$ 9dditionall!, the proteins
can be produced within the cells of the material that carries the bacteria$ 1or
example, the proteins can be produced in the grains of material in animal foods$
6n chic2en feed, the proteins can be expressed in the petals or seeds of the
marigold flower and supplied as marigold meal &that is supplied for its lutein'
which can be added as a feed additive$ 9 number of cereal grains could be
transformed so that the protein was expressed in the seed$ The transgenic corn,
so!bean, canola, peanut, oat, wheat, barle!, rice, sugarbeet, cotton, or tobacco
seed can have the protein expressed therein$ Thus, when the grain is prepared
for animal feed or for human food products, the protein is released from the
seed and is active in decreasing the bacteria present in the grain or feed
product, in the animal that is fed the feed product, and in the meat of animals
fed the feed product$ The proteins can also be placed in soaps, face, and hand
cleansers, as an antimicrobial, and in surface cleansers to decrease bacterial
contamination$
Transgenic corn, so!bean, canola, peanut, oat, wheat, barle!, rice, and other
transformable fruits, vegetables, and plants can have the protein expressed in
tissue other then the seed$ Thus, when the fruit, vegetable, or other plant tissue
or forage is prepared for feeding, the bacteria are decreased$ This is particularl!
useful for the preparation of silage from corn and sorghum$
9dditionall!, the present invention has usefulness in the pharmaceutical field
and the fields of veterinar! science$ learl!, if the bacteria are present in the
digestive s!stem, the mammal can ingest the protein to decrease the bacteria
present$ (ecause man! bacteria are in the gut, b! encapsulating the protein
material or forming the protein in material that is not bro2en down until it
reaches the gut, the protein material can be useful as a method of decreasing the
bacteria in the human or animal digestive s!stem$ 9lternativel!, the protein
could be inGected directl! into the mammalsI intestines or other digestive
organs$
The crude protein extract of the present invention is run on a protein gel to
separate the proteins in the extract$ The proteins are then purified, and each
protein is tested against the listed gram"negative bacteria for the inhibition
effects$ The protein, and<or proteins with the most inhibiting effects, is
seCuenced b! 2nown seCuencing methods$ The methods to do this t!pe of
screening experiment and the procedures involving gene seCuencing and vector
production are clearl! outlined in urrent Protocols in %olecular (iolog!,
published b! +ohn Bile! and Sons, ;ew Hor2 &,--5'$ This manual, or the short
protocols of this manual, can be$ found in most biotechnolog! labs$
Transformation %ethods""are means for integrating new genetic coding
seCuences into the target organismIs genome b! the incorporation of these
seCuences into an organism through manIs assistance$
Using the seCuence of the protein, the 8;9 encoding for such protein could be
isolated and used, via 2nown procedures, to transform a suitable host organism
such that the protein is produced b! the recombinant host in commerciall!
useful amounts$ The protein encoding 8;9 could be reverse"engineered using
codons that are acceptable and recogni)able to the host$ 9lternativel!, the gene
could be isolated b! screening nucleic acid libraries of species which produce
the protein$ 5ligonucleotide probes that are complementar! to a pol!nucleotide
encoding a portion of the protein, for example, a ;"terminus seCuence, can be
emplo!ed to locate the gene$ Probes can be emplo!ed in a 2nown manner to
screen a genomic or c8;9 encoding librar! or to s!nthesis pol!merase chain
reaction &P@' probes for the amplification of the c8;9 encoding for isolates
from an @;9 which translated into the protein of the present invention$ Such
c8;9 could then be cloned into a suitable expression vector for the selected
host and transformed into a host organism$ 5ne of the t!pical host organisms is
#$ coli$ This is not a particularl! useful host for the purposes of this invention
because the protein inhibits the host$ Thus, the host would have to be selected
to be capable of producing the protein without the protein harming the host$
The vector for transformation would preferabl! comprise a nucleotide seCuence
that corresponds to the protein amino acid seCuence that ma! be optimi)ed for
the selected host$ The vector would also be designed with regard to codon
selection, the initiation of translation, the promoter, and the targeting
seCuences, if needed, to maximi)e the expression of recoverable amounts of the
protein$ =ectors for hosts such as plants, algae, insects, animals, !easts, fungi,
bacteria, and humans are commerciall! available from companies such as
;ovagon, and a number of other sources$ =ectors for different hosts are
described in the in urrent Protocols 6n %olecular (iolog!$
There are a large number of 2nown methods to transform plants$ 0owever,
certain t!pes of plants are more amenable to transformation than are others$
Tobacco is a readil! transformable plant, and its transformation is well"
published$ %ost dicots can be transformed b! the methods used to transform
tobacco$ %onocots are transformed b! different methods which are also
widel!"published, and, thus, the basic steps of transforming plants, including
monocots, are 2nown in the art$
These steps are concisel! outlined in U$S$ Pat$ ;o$ 5,.8.,-56 A1ertile
Transgenic *ea ma!s Plants omprising 0eterologous 8;9 #ncoding (acillus
Thuringiensis #ndotoxinA, issued +an$ ,6, ,--6, and in U$S$ Pat$ ;o$ 5,.8-,5/0
AProcess of Producing 1ertile *ea ma!s Plants and Progen! omprising a :ene
#ncoding Phosphinothricin 9cet!l TransferaseA, issued 1eb$ 6, ,--6$
Plant cells, such as mai)e, can be transformed b! a number of different
techniCues$ Some of these techniCues, which have been reported on and are
2nown in the art, include mai)e pollen transformation &see, U$S$ Pat$ ;o$
5,,33,0,0, Universit! of Toledo &,--4''? biolistic gun technolog! &see, U$S$
Pat$ ;o$ 5,.8.,-56'? whis2ers technolog! &see, U$S$ Pat$ ;os$ 5,.6.,365 and
5,40/,5/4'? electroporation &see, P#: on %ai)e'? 9grobacterium &see, ,--6
article on transformation of mai)e cells in ;ature (iotechnolog!, =olume ,.,
+une ,--6'? and numerous other methods which ma! have slightl! lower
efficienc! rates than those listed$
Some of these methods reCuire specific t!pes of cells, and other methods can be
practiced on an! number of cell t!pes$ The use of pollen, cot!ledons,
meristems, and ovum as the target tissue can eliminate the need for extensive
tissue culture wor2$ 0owever, the present state of the technolog! does not
provide ver! efficient use of some of this material$
:enerall!, cells derived from meristematic tissue are useful for transformation
as the! are ver! regenerable$ *!gotic embr!os can also be used$ The method of
transformation of meristematic cells of cereal is taught in the PT application
B5-6<0.4-/$ 9n! of the various cell lines, tissues, plants, and plant parts can
be, and have been, transformed b! those having 2nowledge in the art$ %ethods
of preparing callus from various plants are well"2nown in the art, and specific
methods are detailed in patents and references used b! those s2illed in the art$
ultures can be initiated from most of the above"identified tissue$ The onl! true
reCuirement of the transforming material is that it can form a transformed plant$
The 8;9 used for transformation of these plants clearl! ma! be circular, linear,
double, or single stranded$ Usuall!, the 8;9 is in the form of a plasmid$ The
plasmid usuall! contains regulator! and<or targeting seCuences which assist the
expression of the gene in the plant$ The methods of forming plasmids for
transformation are 2nown in the art$ Plasmid components can include such
items as leader seCuences, transit pol!peptides, promoters, terminators, genes,
multiple gene copies, introns, and mar2er genes$ The structures of the gene
orientations can be sense, antisense, partial antisense, or partial sense$
The regulator! promoters emplo!ed can be constitutive, such as a%v45S
&usuall! for dicots' and pol!ubiCuitin for monocots, or tissue specific
promoters such as 9( promoters$ The prior art includes, but is not limited to,
octopine s!nthase, nopaline s!nthase, a%v,-S, and mannopine s!nthase
promoters$ These regulator! seCuences can be combined with introns,
terminators, enhancers, leader seCuences, and the li2e, in the material used for
transformation$
The isolated 8;9 is then transformed into the plant$ The improvements in
transformation technolog! are beginning to eliminate the need to regenerate
plants from cells$ Since ,-86, the transformation of pollen has been published,
and recentl!, the transformation of plant meristems have been published$ The
transformation of ovum, pollen, and seedlings meristem greatl! reduce the
difficulties associated with cell regeneration of different plants or genot!pes
within a plant$ 8uncan, from at least ,-85",-88, produced literature on plant
regeneration from callus$ Somatic embr!ogenesis has been performed on
various mai)e tissue which was once considered unusable for this purpose$ The
prior art clearl! teaches the regeneration of plants from various monocot and
dicot tissues$
The most common method of transformation is referred to as gunning or
microproGectile bombardment$ This biolistic process shoots small, gold"coated
particles coated with 8;9 into the transformable material$ TechniCues for
gunning 8;9 into cells, tissue, callus, embr!os, and the li2e, are well"2nown
in the prior art$
9fter the transformation of the plant material is complete, the next step is
identif!ing the cells or material which has been transformed$ 6n some cases, a
screenable mar2er is emplo!ed, such as the beta"glucuronidase gene of the uida
locus of #$ coli$ Then, the transformed cells expressing the colored protein are
selected for either regeneration or further use$ 6n man! cases, the transformed
material is identified b! a selectable mar2er$ The putativel! transformed
material is exposed to a toxic agent at var!ing concentrations$ The cells which
are not transformed with the selectable mar2er that provides resistance to this
toxic agent die$ ells or tissues containing the resistant selectable mar2er
generall! proliferate$ 6t has been noted that although selectable mar2ers protect
the cells from some of the toxic affects of the herbicide or antibiotic, the cells
ma! still be slightl! affected b! the toxic agent b! having slower growth rates$
6f the transformed material was cellular, then these cells are regenerated into
plants$ The plants created from either the transformation process or the
regeneration process, or crossed to either of such plants or a progen! of such
plants, are transgenic plants$
The protein of the present invention was extracted from the plant seeds via the
following process7
#J9%PF# ,
Preparation of Protein #xtract from %arigold and Papri2a Seeds
9Cueous extraction was followed b! ammonium sulfate precipitation in the
interval of 40D to 30D relative saturation, heat precipitation at 80E $, and
dial!sis for 4 da!s$ The detailed methods are described below$
1ive hundred grams of marigold seeds &Tagetes erecta' or /50 g of papri2a
seeds &apsicum sp$' were ground in a coffee mill and extracted for / hours in
/$0 liters &marigold' or ,$0 liters &papri2a' of extraction buffer at .E $ The
extraction buffer contained ,0$0 m% ;a0
/
P5
.
, ,5$0 m% ;a
/
0P0
.
, ,00$0
m% >l, /$0 m% #8T9 &eth!lenediaminetetraacetic acid' disodium salt, /$0
m% thiourea and ,$0 m% P%S1 &phen!lmeth!lsulfon!l fluoride' &dissolved in
%e50'$ The homogenate was sCuee)ed through cheesecloth and centrifuged
for 40 min$ at 3000K g$ The pellet was discarded, and the supernatant was
retained$ The supernatant was brought to 40D relative saturation with
ammonium sulfate and precipitated overnight$ The precipitate was removed b!
centrifugation at 3000K g for 40 minutes$ The supernatant was adGusted to 30D
relative saturation with ammonium sulfate and precipitated overnight$ The
precipitate was collected b! centrifugation at 3000K g for 40 min$ The pellet
was re"suspended in .00$0 ml @$5$ &reverse osmosis' water and heat
precipitated in a water bath for 40 minutes at 80E $ The precipitate was
collected b! centrifugation at 3000K g, and the supernatant was dial!)ed
against @$5$ water for 4 da!s using cellulose ester dial!sis tubing with a
molecular weight cut"off of ,000 daltons &SpectralPor, Spectrum, US9'$ The
water was changed 4 times on da! ,, and once each da! for da!s / and 4$ The
dial!)ed supernatant was then fro)en to "60E $ and free)e"dried$
#J9%PF# /
9ntibacterial 9ctivit! 9ssa!
This method was repeated for the marigold and papri2a extracts$ 9
spectrophotometrical method was used where the optical densit! of bacterial
cultures was determined when grown in micro"titer plates$
1or appropriate dilution of the protein extracts from marigold and papri2a, 0$,
gram of the protein extract was blended with ,0$0 grams dextrose to achieve a
homogenous, free"flowing mixture$ 5ne gram of the protein extract<dextrose
preparation was then dissolved in /$0 ml of saline solution and seriall! diluted
in sterile saline$ Twent! microliters of the appropriatel! diluted solution were
then added to the inoculated wells of the micro"titer plate to achieve ,0 ppm,
,00 ppm, or ,000 ppm treatment rates$ ;ext, 80 Ll of a /. hour tr!pticase so!
broth &TS(' culture of Salmonella t!phimurium, #$ coli, or Fisteria
monoc!togenes was added to the wells of a micro"titer plate$ Twelve wells per
organism per treatment rate were used and ,/ further wells were used as a
control series$ 1or the controls, sterile saline, instead of the diluted protein
extract, was added to the wells$
The micro"titer plates were then incubated ,8 hours at 43E $, and the optical
densit! of the wells were subseCuentl! determined spectrophotometricall! at
.05<.,0 nm$ 9s a blan2, sterile TS( was used$
#J9%PF# 4
9ntibacterial 9ctivit! of the Protein #xtracts from %arigold and Papri2a
The antibacterial potenc! of the protein extracts from marigold and papri2a
were evaluated against three gram"negative bacteria, Salmonella t!phimurium,
#$ coli, and Fisteria monoc!togenes$ Potenc! was measured as described in
#xample /$ The three bacteria were tested at 4 different treatment levels, ,0
ppm, ,00 ppm and ,000 ppm, and each treatment level was replicated ,/ times$
9fter ,8 hours incubation, growth of bacteria was measured as optical densit!
as described above$ The results, in percentage of the optical densit! of the
control as mean values for the ,/ replicates, are presented in Table ,$ The
studentIs t"test was conducted to test for statistical significance with PM0$0,
indicating statistical significance$
T9(F# ,
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNN
6nhibition of S$ t!phimurum, #$ coli and F$ monoc!togenes b! protein extracts from
marigold and papri2a as D of control Papri2a &D (acterium<Treatment %arigold &D of
ctrl' T"Test of ctrl' T"Test
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNN
Salmonella t!phimurium
,0 ppm --$0 0$3. ,,0$0 M0$0,
,00 ppm 50$0 M0$0,
68$0 M0$0,
,000 ppm 3,$0 M0$0,
33$0 M0$0,
#$ coli
,0 ppm -6$0 0$06 ,08$0 0$,.
,00 ppm .3$0 M0$0,
.,$0 M0$0,
, 000 ppm 6/$0 M0$0,
34$0 M0$0,
Fisteria monoc!togenes
,0 ppm 58$0 M0$0,
54$0 M0$0,
,00 ppm 48$0 M0$0,
.6$0 M0$0,
, 000 ppm 65$0 M0$0,
68$0 M0$0,
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNN
The results of the gramnegative bacteria stud! clearl! show that there is over a
50D bacterial inhibition at the ,00 ppm level$ The data of T9(F# , is
illustrated graphicall! in 16:$ ,$
#J9%PF# .
6solation and Purification of 0eat Stable Protein 1raction &0SP1'
Upon isolation of the heat stable protein fraction &0SP1', microbial inhibitor!
assa!s were performed to assa! the growth of gram"negative bacteria$ These
tests verified the inhibiting effects of the 0SP1$ 5nce the efficac! was
established, the functional protein, or proteins, of interest were isolated from
the 0SP1 fraction$ 9bout ,00 mg of the heat"stable protein fraction dissolved
in 50 mm %#S &p0 6$0' was applied on a To!opearl Sp"550 cation exchange
column &,0K,$6 cm', previousl! eCuilibrated with %#S buffer$ The column was
eluted at 5$0 ml<min using a step gradient elution ranging from ,00 m% to , %
;al$ #ach fraction eluted from the column was anal!)ed at /80 nm for an!
protein activit!$ Those fractions that showed significant presence were then
dial!)ed into neutral 86 water$ Upon completion of dial!sis, the fractions of
interest were concentrated with 60D w<v pol!eth!lene gl!col &P#:' and then
l!ophili)ed$ The dr! protein extracts were then tested using the same assa! that
was used for the 0SP1$ 9n! fractions showing positive inhibition were then
anal!)ed b! electrophoresis using the Pharmacia phastgel s!stem on a 8"/5
gradient gel$ Upon establishing the inhibiting effects of the fractions and the
protein content of each respective fraction through electrophoresis, the
fraction&s' were passed over a Sephacr!l S",00 0@ in phosphate buffer &0$05
%, p0 3$00' to remove an! residual or undesired protein isolates$ The fractions
were then reanal!)ed on the phastgel s!stem using a homologous gel to provide
greater resolution of the purified protein&s' of interest$ The fractions showing
antibacterial activit! were further anal!)ed b! reversed phase chromatograph!$
9bout , mg amounts of pea2 , material from the Sephacr!l column purification
run were loaded onto a silica ,8 column &/5K-4 cm' in eCuilibrium with 0$,D
T19$ The column was eluted at 5 ml<min with a linear gradient of ,50 ml from
0$,D trifluoroacetic acid &T19' to 50D acetonitrile<0$/D T19$ The elute was
monitored for protein b! online measurement of absorption at /,. nm$ 5nce
satisfactor! purification was achieved, and its presence accounted for the
inhibitor! effects of microbial growth, the protein was read! for seCuencing$
#J9%PF# .
6solation of :ram";egative 6nhibiting Protein from %arigolds and Papri2a
%arigolds and papri2a produce the desired proteins in the seed of the plant$ The
present experiment is adapted to increase the production of that protein$ The
inhibition of the gram"negative bacteria b! the protein can be improved b! the
introduction of the gene that encodes for the desired protein into marigold or
papri2a$ 9dditionall!, the protein can be accumulated in larger seeds such as
the tobacco seed$ 9 plasmid, adapted from a plasmid such as p9T,6,6,
9T accession ;o$ .0806, can have the ph!toene deh!drogenase".0
encoding gene removed and the selected gene inserted b! 2nown restriction site
technolog!$ 9lternativel!, other starting plasmid material can be purchased for
this purpose$ The plasmid is characteri)ed b! the abilit! to be maintained in
9grobacteruim tumefaciens, which is used to infect the tobacco or a number of
other dicots$ The plasmid also has the right and left borders of the seCuence of
the T"8;9 and a promoter associated with the 2anam!cin resistance gene in
the presence of that antibiotic$ The plasmid is transformed into 9grobacterium
tumefaciens strain F(9..0. &F5;T#0, 6nc$' according to standard
protocols$ The tobacco leaf disc are transformed with 9grobacteruim using the
method of 0orsch et$al$, Science, //37,//-",/4, &,-85'$ The selectable mar2er
gene gives resistance to the herbicide norflura)on &Sando)? 0$8 micrograms per
milliliter'$ The plant cells that are transformed do not die in the present of the
herbicide$ The transgenic plants are grown from the transformed cells, and the
seeds are harvested$ Then, five hundred grams of the tobacco seed are ground
in a coffee mill and extracted according to the procedure above for / hours in /
liters of extraction buffer at .E $ The plasmid used for transformation can be
improved b! the use of a promoter that targets the seed$ #xamples of seed
promoters are 2nown in the art, such as the mai)e )ein storage promoter$
9lternativel!, the protein can be produced constitutivel! throughout the plant
with the use of the 45S 9%= promoter, for example$ #xamples of geneticall!
modified plants which ma! be used to produce grain or other plant parts which
express the protein include mai)e, so!bean, sunflower, wheat, barle!, sorghum,
canola, peanut, oats, sugar beet, rice, and tobacco$
9 similar transformation of fungi or bacteria can be performed$ The starting
plasmid would include a promoter recogni)ed b! the host and the selectable
mar2er is often an antibiotic$ The protein product is l!sed from the cell$ To
produce large Cuantities a fermentation process for producing the protein can be
used$
9lthough the invention has been described with respect to a preferred
embodiment thereof, it is to be also understood that it is not to be so limited
since changes and modifications can be made therein which are within the full
intended scope of this invention as defined b! the appended claims$