DETAILED ACTION
In application filed on 02/27/2024, Claims 1-20 are pending. The claim set submitted on 02/27/2024 is considered because this is the most recent claim set. Claims 1-20 are considered in the current office 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 .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 02/24/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections
Claims 1, 7 and 13 are objected to because of the following informalities:
Claim 1 recites the limitation “a detector moiety” in line 5 but earlier recited “a detector moiety” in line 4.
It appears that “a detector moiety” in line 5 should be recited as “the detector moiety”.
Appropriate correction is required.
Claim 7 recites the limitation “the device” but earlier recited “the microfluidic device” in Claim 7.
It appears that the limitation “the device” should be recited as “the microfluidic device”.
Appropriate correction is required.
Claim 13 recites the limitation “degree of aggregation” but earlier recited “level of aggregation” in Claim 1.
It appears that the limitation “the device” should be recited as “degree of aggregation”.
Consistent language should be used and for the purpose of expedited examination the claimed “degree of aggregation” is interpreted as “level of aggregation”
Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-20 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more. The claims have been analyzed for eligibility in accordance with their broadest reasonable interpretation. All claims are directed to statutory categories, i.e., a method (Claims 1-20) (Step 1: YES).
Analysis:
Claim 1: Ineligible.
Step 1:
The claim recites a series of steps or acts, including “method for assessing the aggregation propensity of expressed proteins”. Thus, the claim is directed to a method, which is one of the statutory categories of invention (Step 1: YES).
Step 2A Prong 1:
Claim 1 recites “…to determine the level of aggregation of the protein of interest (mental step)”.
Therefore, the claim is directed towards an abstract idea, and more specifically to the abstract idea group of a mental/math processes since claim 1 relates to using a mental process to determine the level of aggregation of the protein of interest.” (Step 2A, Prong 1: Patent Ineligible).
Step 2A, Prong 2:
This judicial exception is not integrated into a practical application.
Once the determination and comparison are done, No further action takes place.
Also the steps of “expressing a protein of interest having a binding tag that upon binding to a detector moiety generates a fluorescent signal; b. contacting with a detector moiety to generate a fluorescent signal upon binding to the tag; and c. measuring the homogeneity of the fluorescent signal generated upon binding of the detector moiety to the binding tag” are recited at a high level of generality that it amounts to mere data gathering (insignificant extra-solution activity). See MPEP 2106.05(g).
Step 2B:
Furthermore, the courts have found that limitations adding insignificant extrasolution activity to the judicial exception, such as mere data gathering in conjunction with a law of nature or abstract idea, are limitations found not to be enough to qualify as ‘significantly more’ when recited in a claim with a judicial exception (see the 2014 Interim Guidance on Patent Subject Matter Eligibility of the Federal Register dated December 16, 2014; and MPEP 2106.05(I)(A)). Note that mere data gathering is not significantly more than the abstract idea. See MPEP 2106.05(g).
Here, there are no additional elements which are significantly more than the abstract idea. The steps of “expressing a protein of interest having a binding tag that upon binding to a detector moiety generates a fluorescent signal; b. contacting with a detector moiety to generate a fluorescent signal upon binding to the tag; and c. measuring the homogeneity of the fluorescent signal generated upon binding of the detector moiety to the binding tag” appears to be well-understood, routine, and conventional (WURC) in the field of bioanalysis, as evidenced by Waldo et al. (US20050221343A1). (Step 2B: NO).
Therefore, Claim 1 is ineligible.
Moreover, Claims 2-20 are rejected by virtue of their dependency on Claim 1.
Also, each of the dependent claims 2-20 do not solve the issues of claim 1.
Claim 12: Ineligible.
Step 2A, Prong One and Prong Two: Claim 12 further presents an abstract ideas the ratio of soluble and insoluble POI is determine” (mental or math step).
Step 2B: The claims do not recite any elements which are significantly more.
Therefore, Claim 12 is ineligible.
Claims 2-11 and 13-20: Ineligible.
Step 2A, Prong One and Prong Two: Claims 2-11 and 13-20 further define the data gathering steps, which appear to be generic and WURC.
Step 2B: The claims do not recite any elements which are significantly more.
Therefore, Claims 2-11 and 13-20 are ineligible.
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 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.
Claims 1-2, 5-6, 12-15 and 18 are rejected under 35 U.S.C. 102 (a) (1) as being anticipated by Waldo et al. (US20050221343A1).
Regarding Claim 1, Waldo teaches a method for assessing the aggregation propensity of expressed proteins comprising:
a. expressing (See Para 0091… cells are engineered to express both (or, all) complementary fragments, one or two of which are fused to the test protein. The fragments may be expressed simultaneously or sequentially, depending upon whether the assay aims to detect (and quantify) total protein expression or only soluble and/or insoluble fractions. ) a protein of interest (referred to as protein of interest (X) [Para 0020]) having a binding tag (referred to as a small GFP fragment (β-strand 11, residues 215-230) [Para 0020]; See Para 0087… lysing bacterial (or other) cells expressing a fusion of a first fluorescent protein tag fragment and a test protein X (e.g. X-GFP S11 or GFP S11-X)) that upon binding to a detector moiety (referred to as The complementary GFP fragment (β-strands 1-10, residues 1-214) that is expressed separately [Para 0020]) generates a fluorescent signal (See Para 0020… A protein of interest (X) is fused to a small GFP fragment (β-strand 11, residues 215-230) via a flexible linker (L). The complementary GFP fragment (β-strands 1-10, residues 1-214) is expressed separately. Neither fragment alone is fluorescent. When mixed, the small and large GFP fragments spontaneously associate, resulting in GFP folding and formation of the fluorophore);
b. contacting with a detector moiety (referred to as the complementary GFP fragment (β-strands 1-10, residues 1-214) that is expressed separately [Para 0020]) to generate a fluorescent signal upon binding to the tag (See Para 0020… Neither fragment alone is fluorescent. When mixed, the small and large GFP fragments spontaneously associate, resulting in GFP folding and formation of the fluorophore); and
c. measuring the homogeneity of the fluorescent signal (See Para 0087…. The presence of detectable fluorescence in the assay provides an indication that the test protein is soluble) generated upon binding of the detector moiety (referred to as The complementary GFP fragment (β-strands 1-10, residues 1-214) that is expressed separately [Para 0020]) to the binding tag (See Para 0087…contacting the lysate with a second complementary fluorescent protein assay fragment) to determine the level of aggregation (See Para 0153…the total expressed protein content was estimated by adding the protein spot optical densities of the soluble (Ds) and the pellet fraction (Dp) and the solubility was defined as S=Ds/(Ds+Dp); See Para 0098…he insoluble protein fraction is quantified by denaturing and refolding the pellet protein, and combining this preparation with the complementary assay fragment, thereby teaching “level of aggregation”) of the protein of interest (See Para 0087…. The presence of detectable fluorescence in the assay provides an indication that the test protein is soluble) (See Para 0079…If the test protein is soluble, the fusion will be soluble, and thus available for complementation with the other fragment of the reporter protein (the “assay fragment”), which is made available to the test protein-tag fusion protein by, for example, expressing it in the same cell, adding it to a lysate of the cell expressing the fusion protein, etc. In contrast, if the test protein is insoluble, or only partially soluble, the test protein will aggregate, thereby “burying” the fused tag fragment, thus rendering the test protein-tag fusion insoluble and inaccessible for complementation with the assay fragment. If complementation occurs, the detectable reporter phenotype will be activated. For example, where a fluorescent protein is used as the reporter, reconstitution of the characteristic beta-barrel structure following self-complementation of the individually expressed fragments permits the formation of the chromophore, thereby emitting detectable fluorescence; See Para 0020…Processes that make the small GFP tag inaccessible, such as misfolding or aggregation, can prevent complementation See Para 0159…In contrast, when solubilized pellets were diluted with fresh buffer prior to the addition of an aliquot of concentrated GFP 1-10 OPT, several of the well-expressed insoluble proteins (i.e., polysulfide reductase and nucleotide diphosphate kinase, Table 3 and FIG. 14) gave no detectable complementation. Likely these proteins had misfolded and aggregated upon dilution, making the GFP 11 M3 tag inaccessible prior to the subsequent addition of the GFP 1-10 OPT moiety).
Regarding Claim 2, Waldo teaches wherein the fluorescent signal (See Para 0023… Fluorescent images of E. coli BL21(DE3) colonies on nitrocellulose membranes co-expressing GFP 1-10 from superfolder GFP (top), or folding reporter GFP (bottom), along with sulfite reductase fused with wild type GFP S11.) is measured using a thin film (referred to a nitrocellulose membranes [Para 0023]).
Regarding Claim 5, Waldo teaches that the fluorescent signal (See Para 0023… Fluorescent images of E. coli BL21(DE3) colonies on nitrocellulose membranes co-expressing GFP 1-10 from superfolder GFP (top), or folding reporter GFP (bottom), along with sulfite reductase fused with wild type GFP S11.) is measured using a low path length cuvette (See Para 0015…the split-GFP system has been used to quantify proteins in multiwell plates, and to monitor protein expression and solubility in living Escherichia coli cells; Examiner submits that under BRI, Yes, multiwell plates can act very similarly to low-path-length cuvettes.).
Regarding Claim 6, Waldo teaches that the protein is expressed in droplets (See Para 0159… 20 μl of target protein soluble fractions of cell lysates were mixed… in a 96 well microplate (Nunc-Immuno™ plate, Nunc, Rochester, N.Y.); Examiner submits that 20 microliters (µL) is roughly the size of a typical droplet).
Regarding Claim 12, Waldo teaches that wherein the ratio of soluble (referred to as The GFP fragment complementation assay fluorescence of soluble (black bars)[ Para 0032]) and insoluble POI (referred to as unfolded pellet fractions (grey bars) using GFP 1-10 OP [Para 0032]) is determined (See Para 0032; Fig. 14… FIG. 14 bar graph shows in vitro protein quantification of eighteen Pyrobaculum test proteins (see supra, Table 3) with C-terminal GFP S11 M3 tags, using the split GFP system. The GFP fragment complementation assay fluorescence of soluble (black bars) and unfolded pellet fractions (grey bars) using GFP 1-10 OPT. SDS-PAGE gel shows the corresponding soluble (S), and pellet fractions (P). Note that protein #8, tartrate dehydratase β-subunit, shows a second lower band at ca. 13 kD.)
Regarding Claim 13, Waldo teaches that wherein the degree of aggregation (See Para 0153…the total expressed protein content was estimated by adding the protein spot optical densities of the soluble (Ds) and the pellet fraction (Dp) and the solubility was defined as S=Ds/(Ds+Dp); See Para 0098…he insoluble protein fraction is quantified by denaturing and refolding the pellet protein, and combining this preparation with the complementary assay fragment, thereby teaching “level of aggregation”) is measured by counting the number of aggregates, the area of the aggregates, the intensity of the aggregates or by using a measurement of dispersion ((See Para 0153…the total expressed protein content was estimated by adding the protein spot optical densities of the soluble (Ds) and the pellet fraction (Dp) and the solubility was defined as S=Ds/(Ds+Dp), thereby teaching “intensity of aggregates”)).
Regarding Claim 14, Waldo teaches that wherein the binding tag (referred to as a small GFP fragment (β-strand 11, residues 215-230) contains four or more amino acids (referred to as a small GFP fragment (β-strand 11, residues 215-230).
Regarding Claim 15, Waldo teaches that wherein the detector moiety (referred to as the complementary GFP fragment (β-strands 1-10, residues 1-214) that is expressed separately [Para 0020]) comprises a component of a fluorescent protein (See Para 0014…Green Fluorescent Protein (GFP)).
Regarding Claim 18, Waldo teaches that wherein the expression is performed for at least 3 hours (See Para 0092… Cell are then induced to express the test protein-tag fragment fusion for a time sufficient to permit expression of the fusion protein e.g., in E. coli, typically about ½ to 3½ hours)) before the detector moiety which binds to the POI is added (See Para 0091…cells are engineered to express both (or, all) complementary fragments, one or two of which are fused to the test protein. The fragments may be expressed simultaneously or sequentially, depending upon whether the assay aims to detect (and quantify) total protein expression or only soluble and/or insoluble fractions).
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.
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 3 is rejected under 35 U.S.C. 103 as being unpatentable over Waldo et al. (US20050221343A1).
Regarding Claim 3, Waldo teaches that the fluorescent signal (See Para 0023… Fluorescent images of E. coli BL21(DE3) colonies on nitrocellulose membranes co-expressing GFP 1-10 from superfolder GFP (top), or folding reporter GFP (bottom), along with sulfite reductase fused with wild type GFP S11.) is measured in a thin film (referred to a nitrocellulose membranes [Para 0023]).
Waldo does not explicitly teach “a capillary”.
However, it is known that cellulose-based membranes (including nitrocellulose, cellulose acetate, and cellulose triacetate) can absolutely be manufactured in the form of a capillary or tube, which are commonly referred to as capillary fibers or hollow fiber membranes, as evidenced by Cormier et al (US5162227A).
It would have been obvious to one having ordinary skill in the art at the time the invention was made to include that the fluorescent signal is measured in a capillary, since it has been held that the configuration was a matter of choice which a person of ordinary skill in the art would have found obvious absent persuasive evidence that the particular configuration claimed was significant. In re Dailey, 357 F.2d 669, 149 USPQ 47 (CCPA 1966).
Claims 4 and 7-9 are rejected under 35 U.S.C. 103 as being unpatentable over Waldo et al. (US20050221343A1) as applied to claim 1 above, and further in view of Crowther et al. (US20160011208A1, submitted in IDS 02/24/2026 as WO2014132053A1).
Regarding Claim 4, Waldo teaches the fluorescent signal (See Para 0023… Fluorescent images of E. coli BL21(DE3) colonies on nitrocellulose membranes co-expressing GFP 1-10 from superfolder GFP (top), or folding reporter GFP (bottom), along with sulfite reductase fused with wild type GFP S11.).
Waldo does not teach that the fluorescent signal is measured using flow cytometry.
In the analogous art of method for detecting the presence of aggregatory seeds of a polypeptide in a sample is provided, Crowther teaches that the fluorescent signal is measured using flow cytometry (See Para 0036… In addition, the process of aggregate formation can monitored over time by quantification of the fluorescence of the retained aggregate (by measuring intrinsic fluorescence, the fluorescence of the products of seeded co-aggregation with fluorescent monomers or by measurements of fluorescence after staining of the emerging aggregate with amyloid dyes such as e.g. thioflavin T and S, Congo Red), e.g. by flow cytometric (FACS) analysis or imaging techniques.).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo to include that the fluorescent signal is measured using flow cytometry, as taught by Crowther for the benefit of determining the subsequent formation of aggregates of the polypeptide, which may comprise determining the presence of aggregates of the polypeptide in a microdroplet (Crowther, Para 0052), allowing for the provision of new and improved diagnostic assays that can accurately detect the presence of clinically relevant species representing a particular conformation of a protein or an aggregate of polypeptides with a particular conformation. Improved assays are also required for screening candidate therapeutics for an ability to modulate clinically relevant processes underlying the aggregation of proteins that cause conformational diseases (Crowther, Para 0005).
Regarding Claim 7, Waldo teaches the droplets (See Para 0159… 20 μl of target protein soluble fractions of cell lysates were mixed… in a 96 well microplate (Nunc-Immuno™ plate, Nunc, Rochester, N.Y.); Examiner submits that 20 microliters (µL) is roughly the size of a typical droplet).
Waldo does not teach that the droplets are on a microfluidic device.
In the analogous art of method for detecting the presence of aggregatory seeds of a polypeptide in a sample is provided, Crowther teaches that the droplets are on a microfluidic device (See Para 0041… A “microdroplet” is small droplet of a sample that can be manipulated using standard microfluidic technology and devices.; See Para 0043… Techniques for the production of microfluidic aqueous droplets are well known in the art).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo to include that the droplets are on a microfluidic device, as taught by Crowther, for the benefit of manipulating sample droplets (Crowther, Para 0041), allowing for the provision of new and improved diagnostic assays that can accurately detect the presence of clinically relevant species representing a particular conformation of a protein or an aggregate of polypeptides with a particular conformation. Improved assays are also required for screening candidate therapeutics for an ability to modulate clinically relevant processes underlying the aggregation of proteins that cause conformational diseases (Crowther, Para 0005).
Regarding Claim 8, the method of claim 7 is obvious over Waldo in view of Crowther.
Waldo does not teach that the microfluidic device contains channels for flowing droplets.
In the analogous art of method for detecting the presence of aggregatory seeds of a polypeptide in a sample is provided, Crowther teaches that the microfluidic device contains channels for flowing droplets (See Para 0100… Suitable gel forming agents include alginate, gelatine and agarose and other gels having a sol phase sufficiently fluid to move through the channels of a microfluidic device; See Para 0041… A “microdroplet” is small droplet of a sample that can be manipulated using standard microfluidic technology and devices.; See Para 0043… Techniques for the production of microfluidic aqueous droplets are well known in the art).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo to include that the microfluidic device contains channels for flowing droplets, as taught by Crowther, for the benefit of having suitable gel forming agents include alginate, gelatine and agarose and other gels having a sol phase sufficiently fluid to move through the channels of a microfluidic device (Crowther, Para 0100), allowing for the provision of new and improved diagnostic assays that can accurately detect the presence of clinically relevant species representing a particular conformation of a protein or an aggregate of polypeptides with a particular conformation. Improved assays are also required for screening candidate therapeutics for an ability to modulate clinically relevant processes underlying the aggregation of proteins that cause conformational diseases (Crowther, Para 0005).
Regarding Claim 9, the method of claim 7 is obvious over Waldo in view of Crowther.
Waldo does not teach that the device is a digital microfluidic device.
In the analogous art of method for detecting the presence of aggregatory seeds of a polypeptide in a sample is provided, Crowther teaches that the device (See Para 0041… A “microdroplet” is small droplet of a sample that can be manipulated using standard microfluidic technology and devices) is a digital microfluidic device (See Para 0155… this is a powerful characteristic of the digital signal that can be generated by microfluidic techniques, thereby teaching “digital microfluidic device”).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo to include that the device is a digital microfluidic device, as taught by Crowther, for the benefit of demonstrating the capability of by microfluidic techniques (Crowther, Para 0155), allowing for the provision of new and improved diagnostic assays that can accurately detect the presence of clinically relevant species representing a particular conformation of a protein or an aggregate of polypeptides with a particular conformation. Improved assays are also required for screening candidate therapeutics for an ability to modulate clinically relevant processes underlying the aggregation of proteins that cause conformational diseases (Crowther, Para 0005).
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Waldo et al. (US20050221343A1) in view of Crowther et al. (US20160011208A1, submitted in IDS 02/24/2026 as WO2014132053A1) as applied to claim 7 above, and further in view of Mutz et al. (US20020064809A1).
Regarding Claim 10, the method of claim 7 is obvious over Waldo in view of Crowther.
The combination of Waldo and Crowther does not teach that the droplets are spread on a surface by increasing the surface energy of the surface.
In the analogous art of a method is provided for acoustically ejecting from a container that is preferably a channel, a plurality of particles or localized volumes that can be single living cells contained in fluid droplets toward sites on a substrate surface or alternatively or in addition thereto into containers or channels for deposition at a target array site or a container or channel by acoustic ejection, Mutz teaches that the droplets are spread on a surface by increasing the surface energy of the surface (See Para 0080… Once the droplet 63 contacts the substrate surface 51, the droplet modifies an area of the substrate surface to result in an increase or decrease in the surface energy of the area with respect to deposited fluids.).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo and Crowther to include that the droplets are spread on a surface by increasing the surface energy of the surface, as taught by Mutz for the benefit of facilitating the surface modification of the substrate surface (Mutz, Para 0080), allowing for the provision of a method and system capable of sorting a large range of particle sizes without requiring changing the flow tip or addressing other particle size predicated fluidic parameters. Indeed, a need exists for cell sorting methods and systems which do not require such flow tips to eliminate the potential for clogging (Mutz, Para 0016).
Claim 11 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Waldo et al. (US20050221343A1) as applied to claim 1 above, and further in view of Swartz et al. (US20100063258A1).
Regarding Claim 11, Waldo does not teach that the protein is expressed in a cell-free protein synthesis system.
In the analogous art of a method is provided for the cell-free synthesis of the fusion protein, Swartz teaches that the protein is expressed in a cell-free protein synthesis system (See Para 0078… The fusion proteins with and without the Im9 linker were expressed in the cell-free protein synthesis system).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo to include that the protein is expressed in a cell-free protein synthesis system, as taught by Swartz for the benefit of expressing proteins in the cell-free protein synthesis system (Swartz, Para 0078), allowing for the provision of a method that eliminates the requirement to maintain cell viability and allows direct control of various parameters to optimize the synthesis/folding of target proteins. Of particular interest is the synthesis of multi-domain proteins. The present invention provides linkers that are useful in these systems (Swartz, Para 0010).
Regarding Claim 19, Waldo does not teach wherein the expression is performed using cell- free lysates or using assembled components for transcription and translation in a system of purified recombinant elements (PURE).
In the analogous art of a method is provided for the cell-free synthesis of the fusion protein, Swartz teaches wherein the expression is performed using cell- free lysates or using assembled components (See Para 0010… Unlike in vivo gene expression, cell-free protein synthesis uses isolated translational machinery instead of entire cells.) for transcription and translation in a system of purified recombinant elements (PURE) (See Para 0044… Alternatively, for purposes of cell-free expression the construct may include those elements required for transcription and translation of the desired polypeptide, but may not include such elements as an origin of replication, selectable marker, etc.; See Para 0059… In some embodiments of the invention, the fusion protein is produced by cell-free, or in vitro synthesis, in a reaction mix comprising biological extracts and/or defined reagents; See Para 0037… Typically, such substitutions may occur in the polypeptide loops connecting the secondary structural motifs (such as alpha-helical coils) and may introduce, for example, short polypeptides recognized for purification purposes.).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo to include that wherein the expression is performed using cell- free lysates or using assembled components for transcription and translation in a system of purified recombinant elements (PURE), as taught by Swartz for the benefit of expressing proteins in the cell-free protein synthesis system (Swartz, Para 0078), allowing for the provision of a method that eliminates the requirement to maintain cell viability and allows direct control of various parameters to optimize the synthesis/folding of target proteins. Of particular interest is the synthesis of multi-domain proteins. The present invention provides linkers that are useful in these systems (Swartz, Para 0010).
Claims 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Waldo et al. (US20050221343A1) as applied to claim 15 above, and further in view of Reed et al. (US20210364527A1).
Regarding Claim 16, Waldo teaches that wherein the detector moiety (referred to as the complementary GFP fragment (β-strands 1-10, residues 1-214) that is expressed separately [Para 0020]) comprises a component of a fluorescent protein (See Para 0014…Green Fluorescent Protein (GFP)).
Waldo does not teach a further solubility enhancer selected from: Fasciola hepatica 8-kDa antigen (Fh8), Maltose-binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), Small ubiquitin-like modifier (SUMO), Glutathione-S-transferase (GST)…
In the analogous art of methods of identifying and sequencing proteins, polypeptides, and amino acids, and compositions useful for the same, Reed teaches a further solubility enhancer selected from: Fasciola hepatica 8-kDa antigen (Fh8), Maltose-binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), Small ubiquitin-like modifier (SUMO), Glutathione-S-transferase (GST)… (See Para 0172… Examples of tag proteins include, without limitation, Fasciola hepatica 8-kDa antigen (Fh8), Maltose-binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), Small ubiquitin-like modifier (SUMO), Glutathione-S-transferase (GST))…)
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo to include that a solubility enhancer selected from: Fasciola hepatica 8-kDa antigen (Fh8), Maltose-binding protein (MBP), N-utilization substance (NusA), Thioredoxin (Trx), Small ubiquitin-like modifier (SUMO), Glutathione-S-transferase (GST)…, as taught by Reed for the benefit of providing a protein tag (Reed, Para 0172), allowing for the provision of amino acid recognition molecules, such as amino acid binding proteins and fusion polypeptides thereof. In some aspects, the application provides amino acid recognition molecules comprising a shielding element that enhances photostability in polypeptide sequencing reactions (Reed, Abstract).
Regarding Claim 17, Waldo teaches that wherein the protein of interest has a ccGFP11 tag (See Para 0014… test proteins are fused to a sixteen amino acid fragment of GFP (β-strand 11, amino acids 215-230) and the detector moiety comprises ccGFP1-1o (See Para 0014… complementary GFP fragment (β-strands 1 through 10, amino acids 1-214) is added,) .
Waldo does not teach “MBP”.
In the analogous art of methods of identifying and sequencing proteins, polypeptides, and amino acids, and compositions useful for the same, Reed teaches “MBP” (See Para 0172…Maltose-binding protein (MBP) …)
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo to include “MBP”, as taught by Reed for the benefit of providing a protein tag (Reed, Para 0172), allowing for the provision of amino acid recognition molecules, such as amino acid binding proteins and fusion polypeptides thereof. In some aspects, the application provides amino acid recognition molecules comprising a shielding element that enhances photostability in polypeptide sequencing reactions (Reed, Abstract).
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Waldo et al. (US20050221343A1) as applied to claim 1 above, and further in view of Chen et al. (US20230092310A1).
Regarding Claim 20, the method of claim 9 is obvious over Waldo in view of Crowther.
The combination of Waldo and Crowther does not teach that the digital microfluidic device comprises an oil-filled or humidified gaseous environment, wherein the humidified gaseous environment is achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs.
In the analogous art of a method for the cell-free expression of peptides or proteins in a liquid filled digital microfluidic device. The droplets having the components required for cell-free protein expression can be manipulated by electrokinesis in order to enhance levels of protein expression in the droplets, Chen teaches that the digital microfluidic device comprises an oil-filled or humidified gaseous environment, wherein the humidified gaseous environment is achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs (See Para 0016… The oil in the device can be any water immiscible liquid. The oil can be mineral oil, silicone oil, an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The oil can be oxygenated prior to or during the expression process. Alternatively, the device can be an air-filled device where droplets containing cell-free protein synthesis reagents are rapidly moved into position and fixed into an array under a humidified gas to prevent evaporation. Humidification can be achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs. Additionally, humidification can be achieved by connecting an aqueous reservoir to an enclosed or sealed digital microfluidic device. The aqueous reservoir can have a defined temperature or solute concentration in order to provide specific relative humidities (e.g., a saturated potassium sulfate solution at 30° C.).).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Waldo and Crowther to include that the digital microfluidic device comprises an oil-filled or humidified gaseous environment, wherein the humidified gaseous environment is achieved by enclosing or sealing the digital microfluidic device and providing on-board reagent reservoirs, as taught by Chen for the benefit of moving the droplet using any means of electrokinesis (Chen , Para 0015), allowing for the provision of performing CPFS in microfluidic devices (Chen, Para 0012).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Cormier et al. (US5162227A) teaches the use of nitrocellulose tube (See Col. 16, lines 52-23).
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/OYELEYE ALEXANDER ALABI/ Examiner, Art Unit 1797
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