DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Claim 15 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/3/2025.
Applicant’s election without traverse of Group I (current claims 1, 5-7,10-14, & 16-19) and the species SEQ ID NO: 30 and SEQ ID NO: 7 in the reply filed on 10/3/2025 is acknowledged.
The restriction is made FINAL.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Status of Claims
Claims 1, 5-7, 10-14, & 16-19 are under examination on the merits.
Specification
The disclosure is objected to because of the following informalities:
The description of figure 1 (page 17, line 28-page 18 line 2) describes a color legend for various amino acid mutations in the figure. However, the provided drawings are only in black in white. The description should be amended so that it does not reference features that are not visible in the provided black and white drawings.
Appropriate correction is required.
Claim Objections
Claims 5, 13 & 18 are objected to because of the following informalities:
Claim 5 (line 3): “or combinations thereof” should read --and combinations thereof--. Although treatment of claims reciting alternatives is not governed by the particular format used (e.g., alternatives may be set forth as "a material selected from the group consisting of A, B, and C" or "wherein the material is A, B, or C"), claim 5 is written in Markush format, which would require --and-- in order to present a closed group of options. See MPEP 2117(I) for guidance on Markush formatting.
Claim 13 (line 1): “Curcubits” should read --Cucurbits--. Also “the plant is Cucurbitaceae/Cucurbits” should read --the plant is from the family Cucurbitaceae/Cucurbits--.
Claim 18 (lines 1-2): “a nucleotide sequences” should read --a nucleotide sequence--.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
Improper Dependency
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 18 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
The method of claim 17 requires an expression cassette that expresses the non-native sweet protein. Claim 18 requires that the expression cassette comprises a nucleotide sequence encoding the sweet protein. In order for an expression cassette to express a protein, the expression cassette must comprise a nucleotide sequence encoding the protein, so claim 18 fails to further limit the method of claim 17.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 12-14 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 12 requires a plant wherein a progeny or an ancestor thereof is a source of the genomic transformation event enabling the progeny and the ancestor to produce the sweet protein. It is unclear how the progeny of a plant can be the source of a genomic transformation event found in the plant. Genetic material would be passed from ancestor to progeny. If claim 12 intends that the progeny or ancestor is of a plant other than the plant of claim 7, it is unclear what other plant the progeny or ancestor is in relation to. Claim 12 does not clearly define progeny in a way that would enable progeny to serve as a source of the genomic transformation event, so it is indefinite what is encompassed by claim 12 or dependent claims 13-14.
Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 5-7, 12-14, & 16-19 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 1, 5-7, 12-14 & 19 require a nucleotide sequence encoding a sweet protein having a sequence identity of at least 90% identity to SEQ ID NO: 30. Claims 16-18 are drawn to a method for producing any non-native sweet protein.
Nucleic acids with 90% identity to the 159 nucleotide long SEQ ID NO: 30 would have 15 nucleotide substitutions relative to SEQ ID NO: 30. These nucleic acids encompass those in which every substitution is in a different codon and in which every substitution results in codon that encodes a different amino acid. Thus, nucleic acids with 90% identity to SEQ ID NO: 30 encompass nucleic acids encoding sweet proteins with 15 amino acid substitutions relative to SEQ ID NO: 7. These proteins would have 71.7% identity to SEQ ID NO: 7.
A non-native sweet protein encompasses any sweet protein not found natively within the plant of the method of claims 16-18.
The instant specification describes sweet proteins to include thaumatin, monellin, mabinlin, brazzein, egg white lysozyme, pentadin, and neoculin (page 1, lines 28-32). The specification provides no example sequences for any sweet protein other than brazzein.
The specification describes multiple sequences encoding brazzein, with and without epitope tags, flags, or signal peptides (SEQ ID NOs: 8, 10, 12, 21, 23, 25, 27, 29, 30) or codon optimization (SEQ ID NOs: 14-19), complementary or in the same orientation. Of these, SEQ ID NOs: 8, 10, 12, 14, 21, 25, 27, & 29 comprise sequences with 100% sequence identity to SEQ ID NO: 30. See for example SEQ ID NO: 8 below in first alignment.
The next most similar nucleic acid sequence provided by the instant specification is SEQ ID NO: 15, which has 75.8% identity with SEQ ID NO: 30 and 24 mismatches. See second alignment below. There are no nucleic acid sequences taught by the instant specification that encode a sweet brazzein protein with fewer than 24 mismatches except for sequences that have 100% sequence identity.
US-18-565-025A-8
Sequence 8, US/18565025A
GENERAL INFORMATION
APPLICANT: THE COCA-COLA COMPANY
TITLE OF INVENTION: NOVEL BRAZZEIN PRODUCTION SYSTEM AND METHODS
FILE REFERENCE: 60428.0124USWO
CURRENT APPLICATION NUMBER: US/18/565,025A
CURRENT FILING DATE: 2023-11-28
PRIOR APPLICATION NUMBER: PCT/US22/31322
PRIOR FILING DATE: 2022-05-27
PRIOR APPLICATION NUMBER: 63/194,552
PRIOR FILING DATE: 2021-05-28
NUMBER OF SEQ ID NOS: 42
SEQ ID NO 8
LENGTH: 159
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Non-Epitope Tagged Versions, des-pyrE-Brazzein sequence
Query Match 100.0%; Score 159; Length 159;
Best Local Similarity 100.0%;
Matches 159; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 GATAAGTGTAAGAAGGTTTATGAAAATTATCCTGTTTCTAAGTGTCAACTTGCTAATCAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 159 GATAAGTGTAAGAAGGTTTATGAAAATTATCCTGTTTCTAAGTGTCAACTTGCTAATCAA 100
Qy 61 TGTAATTATGATTGTAAGCTTGATAAGCATGCTAGATCTGGAGAATGTTTTTATGATGAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 99 TGTAATTATGATTGTAAGCTTGATAAGCATGCTAGATCTGGAGAATGTTTTTATGATGAA 40
Qy 121 AAGAGAAATCTTCAATGTATTTGTGATTATTGTGAATAT 159
|||||||||||||||||||||||||||||||||||||||
Db 39 AAGAGAAATCTTCAATGTATTTGTGATTATTGTGAATAT 1
US-18-565-025A-15
Sequence 15, US/18565025A
GENERAL INFORMATION
APPLICANT: THE COCA-COLA COMPANY
TITLE OF INVENTION: NOVEL BRAZZEIN PRODUCTION SYSTEM AND METHODS
FILE REFERENCE: 60428.0124USWO
CURRENT APPLICATION NUMBER: US/18/565,025A
CURRENT FILING DATE: 2023-11-28
PRIOR APPLICATION NUMBER: PCT/US22/31322
PRIOR FILING DATE: 2022-05-27
PRIOR APPLICATION NUMBER: 63/194,552
PRIOR FILING DATE: 2021-05-28
NUMBER OF SEQ ID NOS: 42
SEQ ID NO 15
LENGTH: 183
TYPE: DNA
ORGANISM: Artificial Sequence
FEATURE:
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Codon Optimized, des-pyrE-Brazzein-FLAG Maize sequence
Query Match 75.8%; Score 120.6; Length 183;
Best Local Similarity 84.9%;
Matches 135; Conservative 0; Mismatches 24; Indels 0; Gaps 0;
Qy 1 GATAAGTGTAAGAAGGTTTATGAAAATTATCCTGTTTCTAAGTGTCAACTTGCTAATCAA 60
|| |||||||| ||||| ||||| || ||||||||||| || ||||| |||||||| |||
Db 1 GACAAGTGTAAAAAGGTGTATGAGAACTATCCTGTTTCAAAATGTCAGCTTGCTAACCAA 60
Qy 61 TGTAATTATGATTGTAAGCTTGATAAGCATGCTAGATCTGGAGAATGTTTTTATGATGAA 120
|||||||| |||||||| || |||||||| ||||| ||||| ||||| |||||||| |||
Db 61 TGTAATTACGATTGTAAACTGGATAAGCACGCTAGGTCTGGTGAATGCTTTTATGACGAA 120
Qy 121 AAGAGAAATCTTCAATGTATTTGTGATTATTGTGAATAT 159
|| | || ||||||||||| || |||||||| ||||||
Db 121 AAACGCAACCTTCAATGTATATGCGATTATTGCGAATAT 159
The specification also describes brazzein mutants comprising mutations, deletions, alterations, or additions at various residues, including mutations of a sequence of 5 residues (figure 1, page 19, lines 4-17).
Variants of the brazzein protein are known in the art. Assadi-Porter et al (2005) Chem. Senses. 30 (supp 1): i90-i91 (published 1/1/2005) describes brazzein as consisting of one α helix and three anti-parallel β-strands held together by four disulfide bridges (page i90, left column, paragraph 3). Positive charges on the surface of brazzein enhance sweetness and mutations that change these charges to neutral or negative decrease sweetness (page i90, right column, paragraph 3). Variants with changes in H-bonding and less flexibility have reduced sweetness (page i91, left column, paragraph 2-3).
Assadi-Porter et al (2000) Archives of Biochemistry and Biophysics. 376(2): 259-265 (published 5/25/2002, hereafter Assadi-Porter 2000), describes 15 synthetic genes encoding brazzein variants developed through site-directed mutagenesis (page 260, left column, paragraph 3-right column, paragraph 1). Most variants had decreased sweetness, but some increased (table 1). The sweetness of brazzein is affected by protein folding, disulfide bridges, charged residues, the N-terminus, the C-terminus, and residues in the loop containing Arg43 (page 262, right column, paragraph 4-page 264, left column, paragraph 4; figure 4). Assadi-Porter also describes that brazzein is most closely related to plant proteinase inhibitors but lacks three residues in the proteinase specificity loop and has a different pattern of disulfide bonds (page 260, left column, paragraph 1).
The nearest nucleic acid sequence encoding a brazzein protein in the art that does not have 100% sequence identity to instant SEQ ID NO: 30 is a gene used in the construction of an expression vector for recombinant Pichia, with 89% sequence identity (Carlson et al US 2010/112639 A1, published 5/6/2010, see alignment below).
AYA61600
ID AYA61600 standard; DNA; 162 BP.
XX
AC AYA61600;
XX
DT 08-JUL-2010 (first entry)
XX
DE Pentadiplandra brazzeana type III brazzein gene SEQ:4.
XX
KW baking; brazzein; dna cassette; ds; fermentation; food;
KW protein production; protein purification; sweetener;
KW type III brazzein gene; vector.
XX
OS Pentadiplandra brazzeana.
XX
CC PN US2010112639-A1.
XX
CC PD 06-MAY-2010.
XX
CC PF 01-OCT-2009; 2009US-00571743.
XX
PR 06-NOV-2008; 2008US-0111910P.
XX
CC PA (TATE-) TATE & LYLE NORTH AMERICAS LLP.
XX
CC PI Armentrout RW, Carlson A, Ellis TP;
XX
DR WPI; 2010-F06018/32.
XX
CC PT New expression vector for production of brazzein protein by yeast cell,
CC PT comprises cassette containing promoter sequence, secretion signal
CC PT sequence, brazzein protein reading frame sequence, and termination
CC PT sequence.
XX
CC PS Example; SEQ ID NO 4; 19pp; English.
XX
CC The present invention relates to a new expression vector used for the
CC production of brazzein protein by yeast cell. The vector comprises a
CC cassette containing a promoter sequence, a secretion signal sequence, a
CC brazzein protein reading frame sequence, and a termination sequence. Also
CC claimed are: a yeast cell comprising the above-cited expression vector; a
CC recombinant Pichia pastoris cell comprising at least two copies of a gene
CC construct containing pGAP promoter sequence, secretion signal sequence,
CC brazzein type II or type III protein reading frame sequence, and
CC termination sequence; a method for producing enhanced levels of a
CC brazzein protein; a method for enhancing production of a foreign protein
CC in recombinant Pichia; a method for purifying a protein from a yeast
CC fermentation culture. The brazzein protein is useful as sweetener in the
CC baking, beverage and table-top product industries. The present sequence
CC represents a Pentadiplandra brazzeana type III brazzein gene used in the
CC construction of a multi-cassette expression vector for enhanced
CC expression of brazzein as described in an example of the invention.
XX
SQ Sequence 162 BP; 57 A; 23 C; 32 G; 50 T; 0 U; 0 Other;
Query Match 89.3%; Score 142; Length 162;
Best Local Similarity 93.7%;
Matches 148; Conservative 0; Mismatches 10; Indels 0; Gaps 0;
Qy 1 GATAAGTGTAAGAAGGTTTATGAAAATTATCCTGTTTCTAAGTGTCAACTTGCTAATCAA 60
|||||||||||||||||||| |||||||| || ||||||||||| |||||||||||||||
Db 1 GATAAGTGTAAGAAGGTTTACGAAAATTACCCAGTTTCTAAGTGCCAACTTGCTAATCAA 60
Qy 61 TGTAATTATGATTGTAAGCTTGATAAGCATGCTAGATCTGGAGAATGTTTTTATGATGAA 120
|| ||||| ||||| |||||||||||||||||||||||||||||||| ||||| ||||||
Db 61 TGCAATTACGATTGCAAGCTTGATAAGCATGCTAGATCTGGAGAATGCTTTTACGATGAA 120
Qy 121 AAGAGAAATCTTCAATGTATTTGTGATTATTGTGAATA 158
||||||||||||||||||||||||||||| ||||||||
Db 121 AAGAGAAATCTTCAATGTATTTGTGATTACTGTGAATA 158
Given the lack of sequences in the art and the instant specification encoding a brazzein protein with sequence identity of only 90% that of instant SEQ ID NO: 30, one of skill in the art would not recognize that Applicant was in possession of the necessary common attributes or features of the genus in view of the disclosed species.
Other sweet proteins are also described in the art, including brazzein, monellin, thaumatin, mabinlin, and hen egg white lysozyme, and possibly including miraculin and curculin, which taste sweet when combined with sour substances (Temussi (2006) Cell. Mol. Life Sci. 6: 1876-1888, published 7/4/2006, hereafter Temussi; page 1877, right column, paragraph 4). Monellin has no significant similarity to other sweet proteins and belongs to the cystatin superfamily although it lacks the cystatin active site (page 1877, right column, paragraph 6-page 1878, left column, paragraph 1). Thaumatin belongs to the osmotin, thaumatin-like superfamily (page 1878, right column, paragraph 3). Mabinlin shares similarity with seed storage proteins (page 1880, left column, paragraph 1) while hen egg white lysozyme is a lysozyme and shows no significant homology to other sweet proteins (page 1880, left column, paragraph 3). Sweet proteins are structurally diverse, and the instant specification has described only brazzein sequences. Thus, one of skill in the art would not recognize that Applicant was in possession of the necessary common attributes or features of the full scope of the genus of sweet proteins in view of the disclosed species.
Hence, Applicant has not, in fact, described nucleic acids that encode a brazzein protein, nor sweet proteins, over the full scope of the claims, and the specification fails to provide an adequate written description of the claimed invention. Therefore, given the lack of written description in the specification with regard to the structural and functional characteristics of the claimed compositions, Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Claims 10-11, which require that the sweet protein comprises an amino acid sequence having at least 90% identity to instant SEQ ID NO: 7, are described, given that figure 1 and page 19, lines 4-17 describe mutations to the amino acid sequence and some of the effects on sweetness. Mutation of residues 29-33 or 39-43 would constitute 5 amino acid changes to the 53 amino acid-long sequence of SEQ ID NO: 7, or 90% sequence identity.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 16-18 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Evans et al WO 0011196 A1 (published 3/2/2000, hereafter Evans).
Claims 16-18 are drawn to a biosynthetic method for producing a non-native sweet protein.
Evans discloses a method of expressing brazzein in tomato by preparing vector constructs comprising a brazzein gene, fused with a signal peptide or not, under control of a polyubiquitin promoter or polygalacturonase promoter and cloned into a plasmid comprising a terminator (page 19, lines 3-20, figure 10-11). The codons were optimized for tomato expression (page 19, line 17). Evans discloses that the vectors were transferred to Agrobacterium tumefaciens LBA4404, which was used to transform tomato following standard protocols, which were regenerated up to 30 individual plants and grown to maturity (page 19, lines 22-30). This reads on combining a plant with a genomic transformation event that enables the plant to produce a non-native expression of the sweet protein, forming a genetically modified plant and growing and regenerating a population of the genetically modified plant (instant claim 16, lines 3-6). This also reads on preparing plasmids comprising an expression cassette which expresses the sweet protein and comprises nucleotide sequences encoding the protein, transforming a host cell with the plasmids, and transfecting the plant with a plurality of the transformed host cell (instant claims 17-18).
Presence of the transgenic construct in plants was confirmed by PCR and expression of Brazzein was confirmed in one plant with Northern blot (page 19, lines 27-30). Brazzein production in fruits was measured by ELISA and Western blot on total protein extract, which was extracted from a sample of the pericarp of each of the fruit (page 20, lines 1-14). This reads on selecting the genetically modified plants that produce the sweet protein and harvesting the sweet protein (instant claim 16, lines 7-8).
Thus, the method of Evans anticipates claims 16-18.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1, 5, 10, 11, & 16-19 are rejected under 35 U.S.C. 103 as being unpatentable over Evans et al WO 0011196 A1 (published 3/2/2000, hereafter Evans) in view of UniProtKB reference DEF_PENBA (available 7/15/1998).
Claims 1, 5, 10, 11, & 16-19 are drawn to a plant comprising a genomic transformation event which enables the plant to produce a non-native expression or concentration of a sweet protein and a biosynthetic method for producing a non-native sweet protein comprising combining a plant with a genomic transformation event.
The teachings of Evans are presented above. Evans does not teach the expression cassette comprises a nucleotide sequence encoding the sweet protein having a sequence identity of at least 90% to SEQ ID NO: 30. However, Evans does teach that the gene encoding brazzein may be codon optimized. Evans envisions transformed plants of various species, including melon, and the progeny and seeds of such plants comprising a polynucleotide of the invention stably incorporated (page 6, lines 20-30).
UniProtKB reference DEF_PENBA teaches that the protein sequence of brazzein comprises a sequence that can be encoded by a sequence with 100% identity to instant SEQ ID NO: 30 and has 100% sequence identity to instant SEQ ID NO: 7. See alignments below.
DEF_PENBA
ID DEF_PENBA Reviewed; 54 AA.
AC P56552;
DT 15-JUL-1998, integrated into UniProtKB/Swiss-Prot.
DT 15-JUL-1998, sequence version 1.
DT 09-APR-2025, entry version 90.
DE RecName: Full=Defensin-like protein;
DE AltName: Full=Brazzein {ECO:0000303|PubMed:7957951};
OS Pentadiplandra brazzeana.
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Brassicales; Pentadiplandraceae; Pentadiplandra.
OX NCBI_TaxID=43545;
RN [1]
RP PROTEIN SEQUENCE, AND PYROGLUTAMATE FORMATION AT GLN-1.
RC TISSUE=Fruit;
RX PubMed=7957951; DOI=10.1016/0014-5793(94)01184-2;
RA Ming D., Hellekant G.;
RT "Brazzein, a new high-potency thermostable sweet protein from
RT Pentadiplandra brazzeana B.";
RL FEBS Lett. 355:106-108(1994).
RN [2]
RP FUNCTION.
RX PubMed=15118082; DOI=10.1073/pnas.0401567101;
RA Yount N.Y., Yeaman M.R.;
RT "Multidimensional signatures in antimicrobial peptides.";
RL Proc. Natl. Acad. Sci. U.S.A. 101:7363-7368(2004).
RN [3]
RP STRUCTURE BY NMR.
RX PubMed=9628478; DOI=10.1038/nsb0698-427;
RA Caldwell J.E., Abildgaard F., Dzakula Z., Ming D., Hellekant G.,
RA Markley J.L.;
RT "Solution structure of the thermostable sweet-tasting protein brazzein.";
RL Nat. Struct. Biol. 5:427-431(1998).
CC -!- FUNCTION: Taste-modifying protein; sweet-tasting. It is 2000 sweeter
CC than sucrose on a molar basis. {ECO:0000269|PubMed:15118082}.
CC -!- FUNCTION: Has a pH-specific antimicrobial activity against bacteria
CC (B.subtilis, E.coli and S.aureus) and the fungus C.albicans.
CC {ECO:0000269|PubMed:15118082}.
CC -!- SUBUNIT: Monomer.
CC -!- SUBCELLULAR LOCATION: Secreted.
CC -!- SIMILARITY: Belongs to the DEFL family. {ECO:0000305}.
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DR PIR; S51208; S51208.
DR PDB; 1BRZ; NMR; -; A=2-54.
DR PDB; 2BRZ; NMR; -; A=2-54.
DR PDB; 2KGQ; NMR; -; A=2-54.
DR PDB; 2KYQ; NMR; -; A=3-54.
DR PDB; 2LY5; NMR; -; A=2-54.
DR PDB; 2LY6; NMR; -; A=2-54.
DR PDB; 2N66; NMR; -; A=1-54.
DR PDB; 2N69; NMR; -; A=1-54.
DR PDB; 4HE7; X-ray; 1.80 A; A=1-54.
DR PDB; 7W8E; X-ray; 1.34 A; A=1-54.
DR PDB; 7W8H; X-ray; 1.50 A; A/B/C/D/E/F/G/H=1-54.
DR PDBsum; 1BRZ; -.
DR PDBsum; 2BRZ; -.
DR PDBsum; 2KGQ; -.
DR PDBsum; 2KYQ; -.
DR PDBsum; 2LY5; -.
DR PDBsum; 2LY6; -.
DR PDBsum; 2N66; -.
DR PDBsum; 2N69; -.
DR PDBsum; 4HE7; -.
DR PDBsum; 7W8E; -.
DR PDBsum; 7W8H; -.
DR AlphaFoldDB; P56552; -.
DR BMRB; P56552; -.
DR SMR; P56552; -.
DR TCDB; 1.C.45.4.2; the plant defensin (plant defensin) family.
DR EvolutionaryTrace; P56552; -.
DR GO; GO:0005576; C:extracellular region; IEA:UniProtKB-SubCell.
DR GO; GO:0042742; P:defense response to bacterium; IEA:UniProtKB-KW.
DR GO; GO:0050832; P:defense response to fungus; IEA:UniProtKB-KW.
DR GO; GO:0031640; P:killing of cells of another organism; IEA:UniProtKB-KW.
DR Gene3D; 3.30.30.10; Knottin, scorpion toxin-like; 1.
DR InterPro; IPR036574; Scorpion_toxin-like_sf.
DR SUPFAM; SSF57095; Scorpion toxin-like; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antibiotic; Antimicrobial; Direct protein sequencing;
KW Disulfide bond; Fungicide; Pyrrolidone carboxylic acid; Secreted;
KW Taste-modifying protein.
FT CHAIN 1..54
FT /note="Defensin-like protein"
FT /id="PRO_0000221427"
FT MOD_RES 1
FT /note="Pyrrolidone carboxylic acid"
FT /evidence="ECO:0000269|PubMed:7957951"
FT DISULFID 4..52
FT DISULFID 16..37
FT DISULFID 22..47
FT DISULFID 26..49
FT STRAND 4..7
FT /evidence="ECO:0007829|PDB:7W8E"
FT HELIX 13..17
FT /evidence="ECO:0007829|PDB:7W8E"
FT HELIX 21..30
FT /evidence="ECO:0007829|PDB:7W8E"
FT STRAND 33..39
FT /evidence="ECO:0007829|PDB:7W8E"
FT STRAND 41..43
FT /evidence="ECO:0007829|PDB:2BRZ"
FT STRAND 45..50
FT /evidence="ECO:0007829|PDB:7W8E"
SQ SEQUENCE 54 AA; 6498 MW; 5BAB4D7215291252 CRC64;
Length: 54
Score: 315.00 Matches: 53
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 100.0% Indels: 0
Gaps: 0
US-18-565-025A-30 (1-159) x DEF_PENBA (1-54)
Qy 1 GATAAGTGTAAGAAGGTTTATGAAAATTATCCTGTTTCTAAGTGTCAACTTGCTAATCAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 2 AspLysCysLysLysValTyrGluAsnTyrProValSerLysCysGlnLeuAlaAsnGln 21
Qy 61 TGTAATTATGATTGTAAGCTTGATAAGCATGCTAGATCTGGAGAATGTTTTTATGATGAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 22 CysAsnTyrAspCysLysLeuAspLysHisAlaArgSerGlyGluCysPheTyrAspGlu 41
Qy 121 AAGAGAAATCTTCAATGTATTTGTGATTATTGTGAATAT 159
|||||||||||||||||||||||||||||||||||||||
Db 42 LysArgAsnLeuGlnCysIleCysAspTyrCysGluTyr 54
DEF_PENBA
ID DEF_PENBA Reviewed; 54 AA.
AC P56552;
DT 15-JUL-1998, integrated into UniProtKB/Swiss-Prot.
DT 15-JUL-1998, sequence version 1.
DT 09-APR-2025, entry version 90.
DE RecName: Full=Defensin-like protein;
DE AltName: Full=Brazzein {ECO:0000303|PubMed:7957951};
OS Pentadiplandra brazzeana.
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Brassicales; Pentadiplandraceae; Pentadiplandra.
OX NCBI_TaxID=43545;
RN [1]
RP PROTEIN SEQUENCE, AND PYROGLUTAMATE FORMATION AT GLN-1.
RC TISSUE=Fruit;
RX PubMed=7957951; DOI=10.1016/0014-5793(94)01184-2;
RA Ming D., Hellekant G.;
RT "Brazzein, a new high-potency thermostable sweet protein from
RT Pentadiplandra brazzeana B.";
RL FEBS Lett. 355:106-108(1994).
RN [2]
RP FUNCTION.
RX PubMed=15118082; DOI=10.1073/pnas.0401567101;
RA Yount N.Y., Yeaman M.R.;
RT "Multidimensional signatures in antimicrobial peptides.";
RL Proc. Natl. Acad. Sci. U.S.A. 101:7363-7368(2004).
RN [3]
RP STRUCTURE BY NMR.
RX PubMed=9628478; DOI=10.1038/nsb0698-427;
RA Caldwell J.E., Abildgaard F., Dzakula Z., Ming D., Hellekant G.,
RA Markley J.L.;
RT "Solution structure of the thermostable sweet-tasting protein brazzein.";
RL Nat. Struct. Biol. 5:427-431(1998).
CC -!- FUNCTION: Taste-modifying protein; sweet-tasting. It is 2000 sweeter
CC than sucrose on a molar basis. {ECO:0000269|PubMed:15118082}.
CC -!- FUNCTION: Has a pH-specific antimicrobial activity against bacteria
CC (B.subtilis, E.coli and S.aureus) and the fungus C.albicans.
CC {ECO:0000269|PubMed:15118082}.
CC -!- SUBUNIT: Monomer.
CC -!- SUBCELLULAR LOCATION: Secreted.
CC -!- SIMILARITY: Belongs to the DEFL family. {ECO:0000305}.
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DR PIR; S51208; S51208.
DR PDB; 1BRZ; NMR; -; A=2-54.
DR PDB; 2BRZ; NMR; -; A=2-54.
DR PDB; 2KGQ; NMR; -; A=2-54.
DR PDB; 2KYQ; NMR; -; A=3-54.
DR PDB; 2LY5; NMR; -; A=2-54.
DR PDB; 2LY6; NMR; -; A=2-54.
DR PDB; 2N66; NMR; -; A=1-54.
DR PDB; 2N69; NMR; -; A=1-54.
DR PDB; 4HE7; X-ray; 1.80 A; A=1-54.
DR PDB; 7W8E; X-ray; 1.34 A; A=1-54.
DR PDB; 7W8H; X-ray; 1.50 A; A/B/C/D/E/F/G/H=1-54.
DR PDBsum; 1BRZ; -.
DR PDBsum; 2BRZ; -.
DR PDBsum; 2KGQ; -.
DR PDBsum; 2KYQ; -.
DR PDBsum; 2LY5; -.
DR PDBsum; 2LY6; -.
DR PDBsum; 2N66; -.
DR PDBsum; 2N69; -.
DR PDBsum; 4HE7; -.
DR PDBsum; 7W8E; -.
DR PDBsum; 7W8H; -.
DR AlphaFoldDB; P56552; -.
DR BMRB; P56552; -.
DR SMR; P56552; -.
DR TCDB; 1.C.45.4.2; the plant defensin (plant defensin) family.
DR EvolutionaryTrace; P56552; -.
DR GO; GO:0005576; C:extracellular region; IEA:UniProtKB-SubCell.
DR GO; GO:0042742; P:defense response to bacterium; IEA:UniProtKB-KW.
DR GO; GO:0050832; P:defense response to fungus; IEA:UniProtKB-KW.
DR GO; GO:0031640; P:killing of cells of another organism; IEA:UniProtKB-KW.
DR Gene3D; 3.30.30.10; Knottin, scorpion toxin-like; 1.
DR InterPro; IPR036574; Scorpion_toxin-like_sf.
DR SUPFAM; SSF57095; Scorpion toxin-like; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antibiotic; Antimicrobial; Direct protein sequencing;
KW Disulfide bond; Fungicide; Pyrrolidone carboxylic acid; Secreted;
KW Taste-modifying protein.
FT CHAIN 1..54
FT /note="Defensin-like protein"
FT /id="PRO_0000221427"
FT MOD_RES 1
FT /note="Pyrrolidone carboxylic acid"
FT /evidence="ECO:0000269|PubMed:7957951"
FT DISULFID 4..52
FT DISULFID 16..37
FT DISULFID 22..47
FT DISULFID 26..49
FT STRAND 4..7
FT /evidence="ECO:0007829|PDB:7W8E"
FT HELIX 13..17
FT /evidence="ECO:0007829|PDB:7W8E"
FT HELIX 21..30
FT /evidence="ECO:0007829|PDB:7W8E"
FT STRAND 33..39
FT /evidence="ECO:0007829|PDB:7W8E"
FT STRAND 41..43
FT /evidence="ECO:0007829|PDB:2BRZ"
FT STRAND 45..50
FT /evidence="ECO:0007829|PDB:7W8E"
SQ SEQUENCE 54 AA; 6498 MW; 5BAB4D7215291252 CRC64;
Query Match 100.0%; Score 315; Length 54;
Best Local Similarity 100.0%;
Matches 53; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 DKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY 53
|||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 2 DKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY 54
Before the time of filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of Evans to use a nucleotide sequence capable of encoding the brazzein protein taught by UniProtKB reference DEF_PENBA. One of ordinary skill in the art would have been motivated to use a nucleotide sequence codon optimized in another plant species, because although Evans used a sequence codon optimized for tomato, transformation of other species was suggested. One of ordinary skill in the art would have had reasonable expectation of success substituting one nucleic acid sequence for another, because codon optimization was routine prior to the filing of the instant application and the encoded protein would comprise an identical sequence. Thus, claims 16-19 are obvious over Evans and UniProtKB reference DEF_PENBA.
Moreover, the plant comprising a genomic transformation event to produce a non-native expression of a sweet protein comprising an expression cassette comprising a sequence of at least 90% identity to SEQ ID NO: 30, including wherein the expression cassette comprises a signal peptide, promoter, or terminator, would be obvious over Evans and UniProtKB reference DEF_PENBA because the expression cassettes of Evans comprise these regulatory sequences (instant claims 1 & 5). Such a plant would express a sweet protein comprising an amino acid sequence having at least 90% sequence identity to instant SEQ ID NO: 7 (claim 10). Plant parts of the plant, including fruits comprising the sweet protein (instant claim 11), would likewise be obvious.
Claims 1, 5, 10, 11 & 16-19 are obvious over Evans and UniProtKB reference DEF_PENBA.
Claim(s) 6, 7 & 12-13 are rejected under 35 U.S.C. 103 as being unpatentable over Evans and UniProtKB reference DEF_PENBA as applied to claims 1, 5, 10, 11 & 16-19 above, and further in view of Yin et al (2009) Hereditas. 31(6): 663―667 (published June, 2009, hereafter Yin, uploaded with machine translation appended) and in view of NCBI GenBank reference AY262035.1, available 5/24/2003, taken with the evidence of Nunez-Palenius et al (2008) Critical Reviews in Biotechnology. 28: 13-55. (published 10/10/2008, hereafter Nunez-Palenius).
Claims 6, 7 & 12-13 are drawn to the plant comprising a transformation event wherein the regulatory sequences have a sequence identity of at least 70% to instant SEQ ID NOs: 1-6 and 31-36.
The teachings of Evans and UniProtKB reference DEF_PENBA are presented above. Neither teaches a regulatory sequence having an identity of at least 70% to instant SEQ ID NOs: 1-6.
Yin teaches the transformation of tomato with a gene encoding brazzein under the control of the ADP-glucose pyrophosphorylase large subunit 1 promoter from watermelon (page 664, left column, paragraph 4-right column, paragraph 3). Yin teaches that the promoter is expressed in tomato fruit (page 664, left column, paragraph 3 of translation). Yin teaches a motivation to use a fruit specific promoter, because constitutive expression leads to large consumption of nutrients within the plant (page 667, left column, first paragraph of the translation).
Yin teaches that brazzein is high in sweetness, highly water soluble, and can retain sweetness after being treated, making it one of the most promising sweet protein for applications (page 664, left column, paragraph 2 of translation). Yin teaches that sweet protein is both sweet and low in calories, so expressing sweet protein genes can improve flavor (page 666, right column, paragraph 2 of translation).
NCBI GenBank reference AY262035.1 teaches a promoter region for a watermelon ADP-glucose pyrophosphorylase large subunit with 100% sequence identity to 97% of instant SEQ ID NO: 5. See alignment below.
Citrullus lanatus ADP-glucose pyrophosphorylase large subunit (wml1) gene, promoter region
Sequence ID: AY262035.1Length: 1573Number of Matches: 1
Range 1: 276 to 1573GenBankGraphicsNext MatchPrevious Match
Alignment statistics for match #1
Score
Expect
Identities
Gaps
Strand
2398 bits(1298)
0.0
1298/1298(100%)
0/1298(0%)
Plus/Plus
Query 1 GTTAATATGTAAAATCGATAATGCAAAAATATTTTAACTATTTATTTTGAGATGAAATAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 276 GTTAATATGTAAAATCGATAATGCAAAAATATTTTAACTATTTATTTTGAGATGAAATAA 335
Query 61 AGTAGGAGCGATCTTAATCTTCCAATTGTAAAAACATACGTACACATCTAAATTTTATAC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 336 AGTAGGAGCGATCTTAATCTTCCAATTGTAAAAACATACGTACACATCTAAATTTTATAC 395
Query 121 TTAAGGAGGTGTTTGGGCGTGACTTTAAATGGGTGGGGTAAACTATCATAGCTCACTCCA 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 396 TTAAGGAGGTGTTTGGGCGTGACTTTAAATGGGTGGGGTAAACTATCATAGCTCACTCCA 455
Query 181 TGTTTAAGAATGTAGTTATAGTAATTGGTGTTTCCAACTATTATAATTCACAATTAACTA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 456 TGTTTAAGAATGTAGTTATAGTAATTGGTGTTTCCAACTATTATAATTCACAATTAACTA 515
Query 241 CCGTATTACTTGTTACAATATTTACTATTTTCCACCTATCTTCTCTTCCCCTAATTGTTA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 516 CCGTATTACTTGTTACAATATTTACTATTTTCCACCTATCTTCTCTTCCCCTAATTGTTA 575
Query 301 TAGTGTTTATTATTACTTAGACTAAAATAGTATGCACCTTATACATGCAGAAGTAGAATA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 576 TAGTGTTTATTATTACTTAGACTAAAATAGTATGCACCTTATACATGCAGAAGTAGAATA 635
Query 361 GTAATATAGTTTAAAACTACATTGGTCCAGTATCAAGAAGATATCTTAATTTTATTTAGG 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 636 GTAATATAGTTTAAAACTACATTGGTCCAGTATCAAGAAGATATCTTAATTTTATTTAGG 695
Query 421 AATTTAATAATTGGGTGTGGTTTTGTAAACATAAAAATAAAGAAAGCTATAAACAGAGGT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 696 AATTTAATAATTGGGTGTGGTTTTGTAAACATAAAAATAAAGAAAGCTATAAACAGAGGT 755
Query 481 CCTAGGATGCAGATTGGTTACAGAATTCAGTGCCCCAGGAAGTAGCACCAATTTTCATCA 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 756 CCTAGGATGCAGATTGGTTACAGAATTCAGTGCCCCAGGAAGTAGCACCAATTTTCATCA 815
Query 541 TTTATTTAAAAAGTATATTTTGAAGAAATATTATTGTGAAATAGTTTTTGAATTTGTTGC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 816 TTTATTTAAAAAGTATATTTTGAAGAAATATTATTGTGAAATAGTTTTTGAATTTGTTGC 875
Query 601 GTGCTTGGTGGAAGTGGGCAACACAAATACATGGGTGCCCATCCAAGTATTGCCTTGTTT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 876 GTGCTTGGTGGAAGTGGGCAACACAAATACATGGGTGCCCATCCAAGTATTGCCTTGTTT 935
Query 661 GTTTGTTTGTATGCGGCGTAGAAATAGCGGATGATCGTTGGAAATTGAGTTTTGTAGGAA 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 936 GTTTGTTTGTATGCGGCGTAGAAATAGCGGATGATCGTTGGAAATTGAGTTTTGTAGGAA 995
Query 721 TAGCaaaaaaagaaagaaagaaatatgaagaagatgaagatgtaaatgaggaagaagaag 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 996 TAGCAAAAAAAGAAAGAAAGAAATATGAAGAAGATGAAGATGTAAATGAGGAAGAAGAAG 1055
Query 781 aaCCATTTGCTGACATGAATGAACCTTTCCCACTTTCTTGTTTTTTACTATAAATCAATC 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1056 AACCATTTGCTGACATGAATGAACCTTTCCCACTTTCTTGTTTTTTACTATAAATCAATC 1115
Query 841 CTCGTGAATGAAAACGCCTTACATTCACATGCCCATTAAGCATTAATCCCCTTTCTCCAC 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1116 CTCGTGAATGAAAACGCCTTACATTCACATGCCCATTAAGCATTAATCCCCTTTCTCCAC 1175
Query 901 CGCcttcttcatcatcactcacatttgtcactctcttttcctctttctctcttcgtcttc 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1176 CGCCTTCTTCATCATCACTCACATTTGTCACTCTCTTTTCCTCTTTCTCTCTTCGTCTTC 1235
Query 961 ttcCCCCATTTCCAACGCTTCTACCTTTGATTCGTTTCCCCTTTGTAAGTTTTCGATTTC 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1236 TTCCCCCATTTCCAACGCTTCTACCTTTGATTCGTTTCCCCTTTGTAAGTTTTCGATTTC 1295
Query 1021 TTCTGCTTTCCTTTCTGGGATTTCTTATTTGCATCATTTACTTTTCTGGGTGTCTATTAT 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1296 TTCTGCTTTCCTTTCTGGGATTTCTTATTTGCATCATTTACTTTTCTGGGTGTCTATTAT 1355
Query 1081 TTTATTGTATTAGAGCTTTGTCAGATGATTTCTTGTATTTGTTTAGCTACCCCTTTTGCT 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1356 TTTATTGTATTAGAGCTTTGTCAGATGATTTCTTGTATTTGTTTAGCTACCCCTTTTGCT 1415
Query 1141 TTTTTCTGTTCTTGGTGTGATCTGTACTCTATATGGTTGCTTGGATTGGGGCTTTTGCTT 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1416 TTTTTCTGTTCTTGGTGTGATCTGTACTCTATATGGTTGCTTGGATTGGGGCTTTTGCTT 1475
Query 1201 TTTCTTATTGGGATTTGAGCTGGGGGTGGGGCTATTAGATTAGATTGTAATTTGTGCCAT 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1476 TTTCTTATTGGGATTTGAGCTGGGGGTGGGGCTATTAGATTAGATTGTAATTTGTGCCAT 1535
Query 1261 TTCTGATGCAtttttttttttttCCAAGGGAGAGAGTT 1298
||||||||||||||||||||||||||||||||||||||
Sbjct 1536 TTCTGATGCATTTTTTTTTTTTTCCAAGGGAGAGAGTT 1573
Nunez-Palenius provides evidence that melon is in the Cucurbitaceae and methods exist for its transformation (abstract).
Before the filing of the instant application, one of ordinary skill in the art would have been motivated to modify the method and plants taught by Evans to substitute the constitutive promoters with a watermelon AGPL1 promoter as taught by Yin and NCBI GenBank reference AY262035.1. One of ordinary skill in the art would have been motivated to substitute the fruit specific promoter for a constitutive promoter, because constitutive promoters can lead to large consumption of nutrients within the plant. One of ordinary skill in the art would have had reasonable expectation of success, because both methods successfully expressed brazzein in tomato.
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method to transform a melon plant, which is a cucurbit, as suggested by Evans. One of ordinary skill in the art would have been motivated to modify a melon to express brazzein because Yin teaches that brazzein is one of the most promising sweet protein for applications. One of ordinary skill in the art would have had reasonable expectation of success, because melon had been successfully transformed prior to the instant application.
In light of Evans, UniProtKB reference DEF_PENBA, Yin and NCBI GenBank reference AY262035.1, the plant comprising the expression cassette with the regulatory sequence with sequence identity to instant SEQ ID NO: 5, wherein the cassette comprises a promoter operably linked with the nucleotide sequence encoding the protein would be obvious (instant claims 6-7).
Although the meaning of instant claim 12 is indefinite, Evans envisions progeny of the transformed plants. This can read on a plant wherein an ancestor is the source of the genomic transformation event enabling the progeny to produce the sweet protein (instant claim 12).
Claim(s) 14 is rejected under 35 U.S.C. 103 as being unpatentable over Evans, UniProtKB reference DEF_PENBA, Yin, and NCBI GenBank reference AY262035.1 as applied to claims 1, 5-7, 10-13 & 16-19 above, and further in view of Wolf et al (US 2007/0220636 A1, published 9/20/2007, hereafter Wolf).
Claim 14 is drawn to the plant producing a non-native expression of a sweet protein, wherein the plant is watermelon.
The teachings of Evans, UniProtKB reference DEF_PENBA, Yin, and NCBI GenBank reference AY262035.1 are presented above. They do not teach transformation of watermelon.
Wolf teaches a motivation to modify watermelon sweet characteristics, because watermelon fruit quality depends on sweetness (paragraph [0002]). However, elevating total sugar content in watermelon fruit is not desirable because people consume large portions of watermelon; it would be highly advantageous to have watermelon varieties producing fruits that are sweeter yet contain the same amount or fewer calories compared to current watermelon fruit (paragraphs [0007-0008]). The method of Wolf does not involve genetic modification, but Wolf does teach that transformation of watermelon for the addition or deletion of traits is encompassed (paragraph [0020, 0053]).
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of Evans, UniProtKB reference DEF_PENBA, Yin, and NCBI GenBank reference AY262035.1 to substitute watermelon for tomato. One of ordinary skill in the art would have been motivated to introduce a brazzein encoding gene into watermelon in order to create sweeter watermelon without increasing calories. One of ordinary skill in the art would have had reasonable expectation of success, because Wolf teaches that transformation of watermelon is a method to improve sweetness.
Claim(s) 14 is rejected under 35 U.S.C. 103 as being unpatentable over Evans, UniProtKB reference DEF_PENBA, Yin, and NCBI GenBank reference AY262035.1 as applied to claims 1, 5-7, 10-13 & 16-19 above, and further in view of Huang et al US 2023/0263121 A1 (published 8/24/2023 but with an effective filing date of 3/30/2021, prior to the effective filing date of the instant application), hereafter Huang.
The applied reference Huang has a common inventor with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor