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 .
Priority
Applicant’s claim for the benefit and priority of Chinese Patent Application No. 202310117896.0, filed on February 15, 2023 is acknowledged. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
A translation of CN202310117896.0 has not been made of record in accordance with 37 CFR 1.55. When an English language translation of a non-English language foreign application is required, the translation must be that of the certified copy (of the foreign application as filed) submitted together with a statement that the translation of the certified copy is accurate. See MPEP §§ 215 and 216.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-4 and 7-8), in the reply filed on 1/30/2026 is acknowledged. Claims 5-6, drawn to non-elected subject matter have been canceled by the Applicant’s amendment.
Response to Amendment
Applicant’s amendment to the claims filed 1/30/2026 is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Claims 1-4 and 7-8 are pending and have been examined on the merits herein.
Claim Rejections - 35 USC § 112(b)
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 1, 3-4 and 8 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 1 recites “an encoding gene of soybean (Glycine max (Linn.) Merr.)- derived Hb”. There is no special definition for what “derived” means in the context of the claim. Under the B.R.I. of the term, it has been interpreted to mean that the hemoglobin sequence is found in the genome of the indicated soybean species. The term is indefinite because the specification does not clearly define the gene necessary for producing soybean hemoglobin (i.e. leghemoglobin or LegH) and there exist multiple genes or gene products to which the term “derived” could be referring to. Further, it is unclear to what extent derivatives, variants, and truncations are excluded or included by the claim language.
It is established in the art that there exists multiple isoforms of soybean leghemoglobin, and multiple genes which are involved in the production of this hemoglobin-like protein.
Fraser et al. (US PGPub No. 20170342131) teaches that soybean has four leghemoglobin isoforms ([0055]). Specifically, Fraser teaches producing a vector that has a polynucleotide sequence from Glycine max (soybean) encoding LGB2 (FIG. 9, SEQ ID NO:4) ([0273]).
Ahmad et al. (2023. Trends in Food Science & Technology 141 (2023) 104199) teaches that the LGB2 gene encoding LegH C2 from Glycine max, has been successfully synthesized and optimized for expression in Pichia pastoris, which can produce soy LegH C2 with the same amino acid sequence as naturally occurring LegH C2 (pg. 2, right col).
Lee et al. (1983. Nucleic acids research, 11(16), pp.5541-5553) teaches that there are at least four different leghemoglobin (Lb) genes in soybean (Glycine max cv. Prize) (see Fig. 1 and Abstract: “These four genes, including a pseudogene, are present in the same orientation and are arranged in the order: 5'-Lba-Lbc1-LbP-Lbc3-3'. The intergenic regions average 2.5 kb. In addition to this main Lb locus, there are other Lb genes which do not appear to be contiguous to this locus.”, which are shown in Fig. 2). This reference also teaches that “In soybean, there are four major species of leghemoglobin, Lba, Lbcl, Lbc2 and Lbc3 (3), each of which is post-translationally modified into minor species, Lbb, Lbdl, Lbd2 and Lbd3” (pg. 5541).
From the knowledge of the presence of multiple leghemoglobin genes in soybean, and the possibility of different isoforms and derivatives of the leghemoglobin, the instant claims are indefinite because there is no manner in the claim limitations or in the specification to determine which gene sequences are meant to be encompassed or not by the claim limitation.
Therefore, one of ordinary skill in the art would not be reasonably apprised of the scope of the invention and claims 1, 3-4, and 8 are rejected under 112(b) for indefiniteness.
Claims 2 and 7 limit the soybean derived hemoglobin gene to a specific amino acid sequence (which appears to be homologous to that of the LGB2 gene encoding LegH C2) and thus are not rejected herein under 112(b).
Claim Rejections - 35 USC § 103
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1 and 3 are rejected under 35 U.S.C. 103 as being unpatentable over Fraser et al. (US PGPub No. 20170342131).
Fraser teaches methods and compositions for the expression and secretion of heme-containing polypeptides (Abstract), and teaches producing heme-containing polypeptides in recombinant bacterial cells such as Bacillus cells or in recombinant plants or plant cells ([0002]).
Fraser teaches a vector comprising a polynucleotide sequence encoding a heme-containing polypeptide, a signal peptide; and a tag, wherein expression of the polynucleotide sequence in a host cell produces a fusion protein containing the heme-containing polypeptide, the signal peptide, and the tag (e.g., an affinity tag such as a 6-histidine tag or a detectable tag) and genetically modified organisms containing such a vector ([0011]). Fraser suggests culturing such a recombinant bacterium under conditions that allow the heme-containing polypeptide with the signal peptide to be secreted from the bacterium for improved production yield and easier purification of the hemoglobin ([0012]; [0125]-[0132]; [0168]).
Fraser teaches that in some embodiments, the sequence (amino acid and/or nucleic acid) of a leghemoglobin can be a plant leghemoglobin sequence ([0055]). Fraser also teaches that soybean has four leghemoglobin isoforms ([0055]). Specifically, Fraser teaches producing a vector that has a polynucleotide sequence from Glycine max (soybean) encoding LGB2 (FIG. 9, SEQ ID NO:4) ([0273]).
Fraser teaches that suitable bacteria for expression of a polypeptide of the disclosure can be gram negative or gram positive bacteria, including, inter alia, the bacteria can be a species of Escherichia, e.g., E. coli ([0116]).
Fraser teaches that the sequences encoding signal peptides can include, inter alia, those derived from spA, phoA, pelB, ompA, and others ([0081]).
However, Fraser does not explicitly disclose, in a single embodiment, a vector having a polynucleotide sequence from Glycine max (soybean) encoding leghemoglobin and one of the signal peptides from PhoA, PelB, and OmpA, as required of the instant claims.
Regardless, to one of ordinary skill in the art, before the effective filing date of the instant invention, it would have been obvious in view of all of the teachings of Fraser to produce an expression vector (e.g. a cassette) having a sequence for Glycine max (soybean) derived hemoglobin (e.g. leghemoglobin) and any one of these signal peptides, because Fraser describes them as equivalent alternatives for the signal peptide to increase production of plant hemoglobin.
One of ordinary skill in the art would have been motivated by the teachings of Fraser to produce the instantly claimed vector having any one of the signal peptides PhoA, PelB, and OmpA, because they were all disclosed as possible equivalent alternatives, and they are well-known useful signal peptides for secreting proteins from bacteria, particularly from E. coli.
The teachings of Fraser demonstrate the usefulness and benefits of providing a signal peptide when producing leghemoglobin from soybean recombinantly. The selection of a specific signal peptide would have been a matter of judicious selection during routine optimization among the alternatives signal peptides taught in Fraser. See e.g. MPEP § 2144.05.II and MPEP § 2143.I.(E). Thus, one of ordinary would have motivated, with a reasonable expectation of success, to produce an expression cassette, and a recombinant bacteria cell having a plasmid/vector with the expression cassette.
From the teachings of Fraser, it is apparent that there would have been a reasonable expectation of success in combining the teachings therein to arrive at the claimed invention because Fraser uses a sequence of leghemoglobin (i.e. SEQ ID NO:4) that is from soybean (Glycine max) and teaches adding signal peptides for improved production and secretion.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Claim 1, 3, and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Fraser et al. (US PGPub No. 20170342131) as applied to claims 1 and 3 above, and further in view of Wong et al. (WO2022117729).
As described above, the teachings of Fraser make obvious the claimed expression vector cassette comprising a sequence for Glycine max (soybean)-derived hemoglobin (e.g. leghemoglobin) and one of these signal peptides selected from PhoA, PelB, and OmpA, as well as a recombinant cell encoding a vector which comprises the expression cassette.
However, Fraser does not teach that the cell is an E. coli BL21 (DE3) cell.
Wong et al. pertains a method of producing globin polypeptide recombinantly and teaches a vector and a system for recombinant globin polypeptide production as well as a cell comprising a recombinant expression vector (Abstract). Particularly, Wong teaches that the globin polypeptide is selected from the group consisting of a leghemoglobin from a Vigna plant ([001]). Wong also teaches that leghemoglobin from soybean is known in the art ([00312]-[00315]). Regarding the selection of the expression system, Wong teaches that plasmids according to the invention therein were freshly transformed into both E. coli C41(DE3) and E. coli BL21(DE3) ([00270]; Fig. 12).
To one of ordinary skill in the art, prior to the effective filing date of the instant invention, when producing the recombinant cells in order to secrete soybean-derived hemoglobin as taught in Fraser, it would have been obvious to select from among any known E. coli strain, including the known strain BL21(DE3), taught in Wong to be successful for the production of a related plant-derived leghemoglobin protein.
The selection any commonly used bacterial strain for recombinant protein would be well within the knowledge and ability for one with ordinary skill in the art. Such strains are commonly used, and the selection between them amounts to obvious choices, or matters of judicious selection to one having ordinary skill. The instant application does not provide any disclosure regarding the criticality of using the BL21(DE3) strain, nor is there any evidence of unexpected or surprising results when the BL21(DE3) strain is selected.
Thus, the providing of E. coli BL21(DE3) would have been a reasonable selection for producing high amounts of a secreted protein, and one of ordinary skill in the art would have had a reasonable expectation of success, because Wong demonstrates that this strain is used successfully to produce a heterologous hemoglobin protein.
Therefore, the invention of claims 1, 3, and 4 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Claim 1, 3, 4, and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Fraser et al. (US PGPub No. 20170342131) as applied to claims 1 and 3 above, and further in view of Shilling et al. (“Improved designs for pET expression plasmids increase protein production yield in Escherichia coli.” Commun Biol 3, 214 (2020)).
As described above, the teachings of Fraser make obvious the claimed expression vector cassette comprising a sequence for Glycine max (soybean)-derived hemoglobin (e.g. leghemoglobin) and one of these signal peptides selected from PhoA, PelB, and OmpA, as well as a recombinant cell encoding a vector which comprises the expression cassette.
However, Fraser does not teach that the cell is an E. coli BL21 (DE3) cell, as in claim 4, nor that the expression vector is pET-28a, as recited in claim 8.
Shilling et al. pertains to the improvement of pET vectors, including pET28a (Abstract).
In the discussion of what is known to the art, Shilling discusses that the pET series of expression plasmids are widely used for recombinant protein production in Escherichia coli. Further, Shilling teaches that “These expression plasmids support high levels of transcription in strains of Escherichia coli that contain a lysogenized DE3 phage fragment encoding the T7 RNA polymerase and they have become a workhorse for the scientific community” (pg 2, left col). Shilling also teaches that pET28a is the most popular expression plasmid on the market (described in >40,000 published articles), and contains the T7 promoter and an adjacent lac operator sequence that is included to suppress uninduced expression (pg 2, left col, Fig. 1a).
Further, Shilling discusses the use of transformed BL21(DE3) cells for performing protein expression (pg. 7, left col, under “Protein expression and fractionation”).
Therefore, to one of ordinary skill in the art, prior to the effective filing date of the instant invention, when producing a recombinant E. coli cells having expression of the secreted soybean-derived hemoglobin gene, with one of the signal peptides PhoA, PelB, or OmpA, as made obvious from the teachings of Fraser, it would have been prima facie obvious to select the well-known cell line BL21(DE3) and to use the commonly used expression plasmid pET28a, as taught in Shilling, in order to express the hemoglobin protein of interest.
One would have been motivated to select and provide the commonly used bacterial strain BL21(DE3), which is known for its use in recombinant protein production, as taught in Shilling. Further, the selection of the well-known expression plasmid pET28a would have been an obvious selection, within the knowledge and ability for one with ordinary skill in the art.
Such strains and expression vectors are commonly used in the art of recombinant protein expression, and selecting between known strains and vectors amounts to obvious choices or matters of judicious selection to one having ordinary skill.
The instant application does not provide any teaching regarding the criticality of using the E. coli BL21(DE3) strain and the pET28a plasmid, nor is there any evidence of unexpected or surprising results when using the BL21(DE3) strain and the pET28a plasmid. The use of these is common and routine in the art, and successful expression with these is predictable.
Thus, the selection of E. coli BL21(DE3) and the pET28a plasmid, would have been obvious for producing high amounts of a secreted protein, and one of ordinary skill in the art would have had a reasonable expectation of success, because Shilling demonstrates that the pET plasmids, including pET28a, are known in the art, and are commonly used for expressing protein in E. coli. Further, one would predict the success of using E. coli BL21(DE3) with the pET28a because Shilling teaches that the plasmid should be used with a strain having a DE3 fragment.
Therefore, the invention of claims 1, 3, 4, and 8 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date, as evidenced by the cited references, especially in the absence of evidence to the contrary.
Allowable Subject Matter
Claims 2 and 7 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The subject matter in claims 2 and 7 has been found allowable over the prior art of record because an expression vector having at least one of the indicated signal peptides and the hemoglobin gene with the sequence consisting of SEQ ID NO: 4 herein is not disclosed in the art. A nucleotide sequence that is about 60% similar to the instant SEQ ID NO: 4 is taught in Shankar et al. (US PGPub No. 20170349637), which is a polynucleotide sequence from Glycine max (soybean) that encodes LegH/LGB2 ([0155], SEQ ID NO: 5, a Pichia pastoris-Codon-optimized LegH gene). However, there are multiple differences between the two nucleotide sequence, which are due to the codon optimization of the presently claimed sequence. Further, even when comparing the encoded polypeptides, it is noted that the instantly recited peptide is slightly longer than the known LegH peptide taught in the art. The instantly encoded peptide (see instant SEQ ID NO: 8) is three amino acids longer (adding “FLN”) and thus only 97.8 % similar to the wild-type leghemoglobin sequence from Glycine max (see “LGB4_SOYBN. UniProt entry P02236”, included in the PTO-892 accompanying this action). As another example, Fraser et al. (US PGPub No. 20170342131) teaches a polypeptide having SEQ ID NO: 4 therein, which is identical to the polypeptide of the instant SEQ ID NO: 8, except for the last three amino acids, present in the instant sequence and missing in the sequence known in the art.
There is no teachings of a leghemoglobin sequence that consists of the claimed gene sequence herein. Any improved results from fusing this specific sequence with one of the signal peptides would have been unpredictable and thus non-obvious over the prior art.
Conclusion
Claims 1, 3-4, and 8 are rejected herein.
Claims 2 and 7 are objected to, as described above.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ANDREW TERRY MOEHLMAN whose telephone number is (571)270-0990. The examiner can normally be reached M-F 9am-5pm EST.
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/A.T.M./Examiner, Art Unit 1655
/ANAND U DESAI/Supervisory Patent Examiner, Art Unit 1655
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