Patent Application
20250122548
This invention generally relates to protein biochemistry. In alternative embodiments, provided are methods that are reliable and scalable for making disulfide bond rich peptides and proteins. In alternative embodiments, provided are oxidation refolding methods to produce disulfide bond rich peptides and proteins. In alternative embodiments, methods as provided herein can be used to make any disulfide bond-containing proteins, including but not limited to: three finger neurotoxin peptides (such as for example, rec-α-Bungarotoxin (rec-αBtx), rec-α-Cobratoxin (rec-αCTX), κ-Bungarotoxin (rec-κBtx), rec-MTa, rec-hannalgesin, rec-Mambalgin, rec-Slurp, rec-Pate), antibodies and antibody fragments (such as single chain antibody), extracellular domain of viral membrane proteins, cell surface receptors, other disulfide-bond rich toxin peptides (such as dendrotoxin, conotoxin) and the like.
Many proteins or small peptide contain sophisticated disulfide bonds, which drastically increase the stability of the structure. On the other hand, the presence of such bonds makes it very hard to obtain these proteins directly from Escherichia coli, or E. coli.
Snake venom three finger toxin peptides (or three finger proteins or peptides, or TFPs) are such a group of macromolecules that have interesting properties. Some of them can bind nicotinic acetylcholine receptors in the muscle (muscle type nAChR), some of them have analgesic effect, such as mambalgin and hannalgesin, some of them bind to receptors in the neurological system, such as MTa and κ-Bungarotoxin, which bind to α 2B-adrenoceptor and α3β2 nAChR, respectively.
The mammalian genome also encodes a group of three finger toxin-like proteins whose function little is known, such as Slurp, Lynx and Pate.
Due to the complex disulfide bonds system in these toxin peptides, it is usually impossible to obtain correctly folded three-finger toxin peptide directly from E. coli and not efficiently produced from other secretion expression systems, such as Pichia pastoris. There were successful attempts using chemical synthesis, such as muscarinic toxin MT7 and MT and Mambalgin, but this method suffers from the problem of sophisticated synthesizing steps, low productivity, and extremely high costs. As such, a universal, high yield production protocol capable of producing high quality three finger neurotoxins is needed.
In alternative embodiments, provided are methods for producing a disulfide-linked protein, comprising:
In alternative embodiments, the incubating comprises conditions comprising a temperature of about 4° C. or an ice-cold solution, and/or incubating under pressure, optionally by use of compressed air or by use of a nitrogen tank or a reaction vessel.
In alternative embodiments, provided are methods for producing and purifying, or substantially purifying or isolating, a disulfide-linked protein, comprising:
In alternative embodiments of methods as provided herein:
In alternative embodiments, provided are kits or products of manufacture comprising all materials, reagents and ingredients needed to practice methods as provided herein, and optionally also comprising instructions for practicing methods as provided herein.
The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.
Figures are described in detail herein.
Like reference symbols in the various drawings indicate like elements.
In alternative embodiments, provided are methods that are reliable and scalable for making disulfide bond rich peptides and proteins. In alternative embodiments, provided are oxidation refolding methods to produce disulfide bond rich peptides and proteins. In alternative embodiments, methods as provided herein can be used to make any disulfide bond-containing proteins, including but not limited to: three finger neurotoxin peptides (such as for example, rec-α-Bungarotoxin (rec-αBtx), rec-α-Cobratoxin (rec-αCTX), κ-Bungarotoxin (rec-κBtx), rec-MTa, rec-hannalgesin, rec-Mambalgin, rec-Slurp, rec-Pate), antibodies and antibody fragments (such as single chain antibody), extracellular domain of viral membrane proteins, cell surface receptors, other disulfide-bond rich toxin peptides (such as dendrotoxin, conotoxin) and the like, which have wide applications.
The method is exemplified in producing recombinant three finger neurotoxin peptides from E. coli. Traditionally, such three finger neurotoxin peptides were purified from snake venoms, which is sophisticated and generally limited by the scarce source of snake venoms and hence very expensive. The recombinant three finger neurotoxin peptides produced by methods as provided herein were confirmed by either x-ray diffraction structural analysis and or activity test using either in vitro binding assay with known receptors, or by using immunofluorescent staining of known targets on live cells. In alternative embodiments, methods as provided herein provided an attractive alternative source of the disulfide bond rich peptides and proteins, including three finger neurotoxin peptides, which have great application potential both scientifically and commercially.
In alternative embodiments, methods as provided herein have at least three unique aspects:
First, the toxin peptides are expressed without any tag, thus significantly simplifies the purification procedure.
Second, for inclusion body oxidative refolding, we developed a unique protocol using a custom designed oxidation chamber, which also functions as a storage tank (as illustrated in
Third, no redox-pairs such as reduced-oxidized glutathione, or cysteine-cystine are used, only cysteine concentrations are optimized.
Using exemplary methods as provided herein, we have successfully produced recombinant three finger neurotoxin peptides such as rec-α-Bungarotoxin (rec-αBtx), rec-α-Cobratoxin (rec-αCTX), κ-Bungarotoxin (rec-κBtx), rec-MTa, rec-hannalgesin, rec-Mambalgin, rec-Slurp, rec-Pate, and the like, with five of them being structurally validated. As an example, shown here, the structural alignment of natural κBtx aligned with rec-κBtx shows the two aligned perfectly (see
In alternative embodiments, methods as provided herein include use of a specially designed oxidation chamber where the redox potential is monitored, which enabled refolding condition being closely monitored, thus drastically improved the reproducibility of the process and the quality of final product.
In alternative embodiments, methods as provided herein have are carried out in general molecular biology lab and can be scaled up easily. Given the importance of these toxin peptides in biomedical research, methods as provided herein are a significant step-forward in the field.
Finally, our method should in principle be applied to refold other disulfide bond rich proteins, which should be of general interest both scientifically and commercially.
Example Result is illustrated in
Provided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein.
Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections.
As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of”, “substantially all of” or “majority of” encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition.
The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court.
Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.
The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.
Unless stated otherwise in the Examples, all techniques are carried out according to standard protocols.
This example demonstrates that methods as provided herein are effective and can be used to product disulfide bonded peptides and proteins.
Provided herein is a working pipeline for expression, purification of disulfide-bond rich three-finger neurotoxin peptides of snake venom origin, or their homologous protein of mammalian origin, using E. coli as the expression host. With this pipeline, we have successfully obtained high quality recombinant α-Bungarotoxin, k-Bungarotoxin, Hannalgesin, Mambalgin, α-Cobratoxin, MTα, Slurp1, Pate B etc. Milligrams to hundreds of milligrams of recombinant three finger proteins can be obtained within weeks in the lab. The recombinant peptides showed specificity in binding assay and six of them were crystallized and their structures were validated using X-ray protein crystallography.
Our method is different from previous attempts in that, 1. The recombinant toxins were expressed without any fusion tags, thus significantly simplifying the purification procedure and dramatically increasing the quality and the yield. 2. For each toxin, a universal refolding screen protocol was applied to search for refolding conditions. 3. A unique oxidation refolding protocol was carried out to ensure complete disulfide bond formation. Due to the extremely high quality of the recombinant peptides and high yield, our method provides an attractive alternative source of three-finger toxins or toxin-like proteins to their natural counterpart.
Lysis buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 10 mM 2-mercaptoethanol (5 liter for 200 g of bacteria cell pellets)
Solubilization buffer: 50 mM Tris-HCl (pH 9.0), 6 M guanidine-HCl (or 8 M urea), and 5 mM 2-mercaptoethanol (should be freshly prepared).
Refolding screen buffer (see Table 2, below).
Restriction enzymes were from Takara or New England Biolabs. All chemicals were from Sigma-Aldrich unless otherwise stated.
Vector Construction, E. coli Fermentation and Inclusion Body Extraction
Genes encoding the toxin proteins were codon-optimized and synthesized (Integrated DNA Technologies Inc) with NdeI site on the 5′ end and a termination codon (TAA or TAG) at the 3′ end just before the XhoI sites. The genes were inserted into the NdeI and XhoI sites of pET30b (Novagen) and the reconstructed expression vector was transformed into the expression host BL21 (DE3). E. coli cells were fermented either with a home-made 5-liter fermenter or a BIOFLO3000 BIOREACTOR™ (New Brunswick Scientific) and induced for protein expression by adding 0.8 mM IPTG at an Optical density of approximately 18 to 19 and fermented for additional 4 hrs. In alternative embodiments, about 160 g to 400 g of bacteria pellets (wet weight) could be obtained and stored at −20° C. as 50 grams aliquots. To obtain the inclusion bodies, 200 g of bacteria was thawed in 1 liter of lysis buffer supplemented with 2 mg chicken egg lysozyme per gram of bacteria pellets of was then added and mixed well using a bench-top homogenizer (KitchenAid). The mixture was incubated on ice for 1 hr and sheared with the homogenizer at top-speed for 60 s and cooled in the cold room for 15 min, the shearing process was repeated twice until the solution become less sticky, which was then centrifuged at 10,000 g/4° C./15 min. The supernatant was discarded, and the pellets were subjected to a new round of resuspension-shearing-centrifugation process until the pellet became compact. The pellets were finally resuspended in 1 to 2 liters of lysis buffer and aliquot to 20 to 40 50-ml conical tubes, pellet down by centrifugation at 8000 g/10° C./15 min, and stored at −20° C. until use.
To solubilize the I.B., a solubilization buffer containing 50 mM Tris-HCl (pH 8.0), 8 M urea or 6 M guanidine-HCl and 5 mM 2-Mercaptoethanol (2-ME) was used. The choice of the solubilization buffer was based on the solubilization effect and contaminating protein level. Taken α-Bungarotoxin for example, this toxin refolded poorly in the presence of contaminating proteins and its I.B. was solubilized well with a solubilization buffer containing 8 M urea. So, after solubilization with 50 mM Tris base, 8 M urea, and 5 mM 2-mercaptoethanol, and centrifuged for 28,000 g/10 min/4° C. to get rid of insoluble bacteria debris, the pH of the supernatant was adjusted to 8.5 with concentrated HCl solution and further absorbed with Q Sepharose FF media (2 ml of solution/ml of Q media) equilibrated with 50 mM Tris-HCl (pH 8.5), 8 M urea. Attention should be paid to avoid using high concentration of reducing chemical reagents (like 100 mM of 2-mercaptoethanol) at the solubilization stage, which will lead to low refolding efficiency.
After absorption, the I.B. was ready for refolding. For other toxins with higher expression level and more compact inclusion bodies, a solubilization buffer containing 50 mM Tris-HCl (pH 9.0), 6 M guanidine-HCl, 5 mM 2-ME was used.
Refolding condition was optimized with a screening protocol scouting for NaCl concentration (0 or 200 mM), l-cysteine concentration (0-16 mM), 1-arginine (0 or 0.5 M), and detergent, such as NDSB-201 (0 or 0.2 M), etc. Standard refolding trial was made by diluting 200 μl of I.B. solution into 10 ml of refolding screen solution.
After refolding, the solutions were left at 4° C. overnight (more than 24 hr), and concentrated each with Amicon Ultra-15 (Millipore, 3 kDa NMWL) ultrafiltration devices to less than 200 μl. The retention was centrifuged at 18,000 g/4° C. for 15 min and the supernatant analyzed with non-reducing SDS-PAGE. The rest of the concentrated solutions were each divided into three parts and dialyzed against low ionic strength buffer with various pH values, such as 20 mM NaAc (pH 5.0), 20 mM HEPES (pH 7.5, adjusted with NaOH), or 20 mM Tris-HCl (pH 8.0), using home-made micro-dialysis devices (Fiala et al., 2011). Finally, the dialyzed solution was centrifuged at 18,000 g/4° C. for 15 min. The supernatant was analyzed using non-reducing SDS-PAGE for quantifying different refolding species of monomer and multimers.
After the initial screen, an optimized refolding condition was usually determined, based on the yield of monomeric species on non-reducing SDS-PAGE. For preparative refolding, fresh I.B. solution was poured all in once into the ice-cold refolding solution which was stirred rapidly by a magnetic bar throughout the whole process, at a volume ratio of 1:50. The refolded solution was left static over one week at 4° C., and concentrated with a compressed nitrogen-gas (or air) driven ultrafiltration device (350 ml Amicon Stirred Cell, 3 kDa NMWL membrane, Millipore) (coupled with a storage tank in refolding recombinant κ-Bungarotoxin). Typically, the refolded solution was concentrated to a very small volume of several ml, or to ‘dry’, depending on the type of the toxins proteins being refolded, and then filled with refolded protein solution and proceeded to do the ultrafiltration again, this process is repeated back and forth until all the refolded solution was concentrated, which usually takes about 2-4 weeks, during which time cysteine in the solution gradually react to form white cystine crystalline and precipitated out, and could usually clog the ultrafiltration membrane at the end phase of concentration, making the process longer. For some toxins, like recombinant MTα (rec-MTα), Hannalgesin (rec-Hannagesin), mouse Pate B (rec-mPateB), κ-Bungarotoxin (rec-κBtx), α-Bungarotoxin (rec-αBtx), it is better to concentrate to ‘dry’, leaving no noticeable liquid in the ultrafiltration device, which could dramatically increased the purity and quality of the product. For other toxins we tried like recombinant mambalgin-1 (rec-Mambalgin-1), mouse and human Slurp1 (rec-mSlurp1 and rec-hSlurp1), concentrating to dry significantly lowered the final yield. So, trial experiments should be made at this point. The concentrated product was then re-solubilized with a low ionic strength buffer, which was pre-determined in the dialysis experiment. Normally, proteins with isoelectric point (pI) over 7 was re-solubilized in 30 to 50 ml of 50 mM NaAc (pH 5.0), while proteins with pI less than 7 was solubilized in 50 mM Tris-HCl (pH 8.0). The solution was then filtered with a 0.2 μm filter and applied to mono S 5 50 GL column or mono Q 5 50 GL column, based on the pI of the proteins.
Bound proteins were eluted with a linear gradient of NaCl to 1 M. The eluted peaks were again analyzed by non-reducing SDS-PAGE. Those eluted later usually contained contaminating proteins, or species inter-connected by intermolecular disulfide bonds.
For those not concentrated to ‘dry’ but to small volume, an additional dialysis step was usually added, in which the concentrated solution was dialyzed against the low-ionic strength buffer and applied to the ion exchange column. For the proteins we tried, a single, large peak was usually seen using the mono S column (See result), and several large peaks were seen using the mono Q column, in which the target species was usually contained in the first peak. At this stage, the refolded toxin was fairly pure, but for XRD experiments, gel filtration was usually done with a Superdex 75 10 300 GL column (GE Healthcare), to further increase the purity of the product and to buffer-exchange to 200 mM ammonium acetate (pH 7° C.).
5 μg of HAP peptide (Harel et al., 2001; Kudryavtsev et al., 2020) were mixed with 5 μg of recombinant respectively, incubated at room temperature for 15, and run on a 15% native PAGE gel with 50 mM NaAc (pH 5.0) at 120 v/60 min/4° C. For the binding assay with the nicotinic acetylcholine receptors, 5 μg of rec-αCTX, rec-Hannalgesin, or α-Cobratoxin (αCtx) (Sigma-Aldrich, C6903) was mixed with 5 μg of recombinant the extracellular domain of the al subunit of muscle type nicotinic acetylcholine receptor (α1ECD) (Dellisanti et al., 2007; Yao et al., 2002), incubated on ice for 15 min and run on 12% native gel (standard discontinuous PAGE gel (without SDS), 6% for top layer and 10% for bottom layer with Tris-Glycine buffer (pH 8.3, without SDS) as the running buffer at 120 v/90 min/4° C. Gels were stained with coomassie brilliant blue as described (Wittig and Schägger, 2005).
Labeling of Rec-mPate B with Fluorescence Dye and Visualization of Binding of Rec-mPate B to the Mouse Spermatozoa
rec-mPate B was labeled with NHS-rhodamine according to the manufacturer's recommended protocol. Briefly, 25 μl of rec-mPate B solubilized in PBS (pH 7.4) at 27.2 mg/ml was mixed with 20 mM HEPES (pH 7), 4.13 μl of 18.9 mM NHS-Rhodamine DMSO solution (ThermoFisher) and incubated at room temperature for 60 min, and dialyzed exhaustively against 20 mM HEPES, 0.15 M NaCl. Mouse spermatozoa was obtained as described, and was mixed with 1:1000 dilution of the Rhodamine labeled rec-mPate B, washed three times with PBS, and observed under a laser confocal fluorescence microscope.
X-Ray Protein Crystal Diffraction Structural Validation of rTFP
Purified toxin proteins were concentrated to 15 to 150 mg/ml with Amicon Ultra-15 and Amicon Ultra-0.5 (3 kDa NMWL) tubes. Sitting drop crystal screening was done using a robotic system (Crystal Gryphon, Art Robbins Instrument). For crystallization of rec-αBtx, rec-αBtx was complexed with HAP peptide (Harel et al., 2001) by mixing at a molar ratio of 1:1.5, incubated at room temperature for 30 min and then diluted 100 fold with 20 mM NaAc, pH 5.0 and applied to mono S column. Bond protein was eluted with linear gradient of NaCl to 1 M and the sharp peak containing the rec-αBtx-HAP complex was collected, pooled and concentrated to about 13 mg/ml, dialyzed against 0.1 M HEPES (pH 7.0) exhaustively at 4° C.
For crystallization of other recombinant three-fingered proteins (rTFPs), purified rTFP were concentrated to about 80 to about 150 mg/ml and screened for crystal growth. Hanging drop method was then done manually to optimize the growth condition, by mixing equal volume of well solution and the toxin protein, and incubating both at 4° C. and 18° C.
Crystals were then harvested under cryo-conditions and X-ray diffraction data of for rec-kBtx·rec-mambalgin 1 and rec-αBtx-HAP complex were collected either with a RIGAKU MICROMAX™-007 home X-ray source coupled with an R-AXIS IV++ image plate. For rec-MTα, X-ray diffraction data was collected at ADVANCED PHOTON SOURCE™ (Argonne National Laboratory, Lemont, IL). The X-ray diffraction data of rec-Hannalgesin and rec-αCTX were collected at Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley, CA).
Data was processed with HKL2000™ (Otwinowski and Minor, 1997) or IMOSFLM™ (Battye et al., 2011), CCP4 suite (Winn et al., 2011), Molecular Replacement, structure build and refinement was done in PHENIX™ (Liebschner et al., 2019) and Coot (Liebschner et al., 2019).
Our Pipeline is Universally Applicable to a Wide Variety of TFPs with High Yield
Our idea is to use E. coli to produce high quality three-fingered proteins (TFPs) of biomedical interests. Our pipeline involved codon optimization of the encoding DNA sequence, recombinant protein expression in E. coli, isolation of I.B., refolding condition scouting, preparative refolding and purification, structural validation with x-ray diffraction and biochemical methods (see exemplary protocol of
It is hard to imagine expressing, refolding rTFP of several kDa using E. coli and achieving milligrams to hundreds of milligrams in a common molecular biology lab. However, with our pipeline, we obtained over one hundred milligrams of rec-MTα, rec-Hannalgesin, rec-αCTX and rec-mPate B, tens of milligrams of rec-mambalgin-1, Slurp1 and milligrams of rec-kBtx and rec-αBtx with only one round of experiment (usually finished within approximately 4 to 5 weeks), which to our knowledge, has never been reported before.
Most Useful Scouting Conditions for Refolding rTFP are Cysteine and Salt Concentration, and pH
For optimized refolding condition for each recombinant neurotoxin, the most critical factors are the concentration of sodium chloride and l-cysteine and pH. L-arginine (see for example, Arakawa et al., 2007; Chen et al., 2008; Tischer et al., 2010; Tsumoto et al., 2004) and NDSB-201 (Luca et al., 2012; Wangkanont et al., 2015), two known supplements which are widely used in inclusion body refolding, even though significantly increased the yield of monomeric species in the screening experiment as reflected by non-reducing SDS-PAGE (
In addition, recombinant three-fingered proteins (rTFPs) refolded with l-arginine usually were hard to crystallize (data not shown). What's more, l-arginine and NDSB-201 are very expensive and not cost-effective in large scale production. Taken together, L-arginine and NDSB-201 are generally not helpful for refolding rTFPs, at least for the rTFPs we attempted.
Normally, rTFPs with high isoelectric point (pI) remained soluble upon challenge with weak acidic solution (such as 20 mM NaAc, pH 5.0), while certain mammalian toxin-like protein, such as Slurp1, remained soluble only in neutral and slight basic solutions. Usually, if the refolded product remains soluble after the dialysis step, and does not contain species with significant inter-chain disulfide bond, as judged by existence of multimeric species on non-reducing SDS-PAGE, it is highly possible that the refolding is successful.
Complete Oxidation is the Key for High Quality rTFPs
It is common to see I.B. refolding protocols in which people dissolve the I.B. with solutions containing high concentration of reducing agents (such as 100 mM β-mercaptoethanol or 2-ME). While these agents are useful in keeping the free cysteine residue in reduced form and it might not be a problem in certain cases, we found 100 mM 2-ME in I.B. solubilization buffers inevitably lead to failed refolding experiments, which was shown by the extremely low yield and formation of multimeric species (Xu et al., 2015), thus should be avoided when solubilizing the I.B. For correct disulfide bonds pairing between the cysteine residues, a classical and widely used approach is the disulfide shuffling or mixed disulfide bond reactions, in which a predefined redox pairs such as a fixed ratio of reduced-glutathione:oxidized-glutathione, or cysteine:cystine are used (“Disulfide bond formation in proteins,” 1984; Okumura et al., 2011; Qin et al., 2015).
In our pipeline, we used a simple, straightforward approach by scouting cysteine concentration in screening refolding conditions, and we noticed that different recombinant three-fingered proteins (rTFPs) had different sensitivity to cysteine concentration in the refolding experiment, see
It is interesting to note this point, since we found that multimeric species, which were generally regarded as incorrectly folded product with wrong pairing of disulfide bonds, were always present in the refolding product and hard to be separated from the correctly folded species using chromatography approaches, such as gel filtration and ion exchange. However, it turned out that ultrafiltration of the refolded product to dry dramatically increased the purity for some rTFPs, see
Recombinant rTFPs Shows Good, Unique Behavior in Gel Filtration Chromatography and Biochemical Assays
We compared the behavior of rec-αBtx, rec-κBtx, rec-mPate B in gel filtration column (SUPERDEX 75 10 300 GL™, GE Healthcare), and found that rec-αBtx behave like a monomer, while rec-κBtx behave like a dimer, see
To test the binding specificities of the recombinant three-fingered proteins (rTFPs), HAP peptide, a known peptide derived from the nicotinic acetylcholine receptor (Harel et al., 2001), was mixed with various rTFPs and separated on a native PAGE gel at pH 5.0. HAP peptide was only able to shift rec-αBungarotoxin and only slightly shift rec-Hannalgesin, but not rec-MTα, rec-mPate B, rec-κBtx, and rec-hSlurp1, see
Structural Comparison of rTFPs Shows Almost the Same Structure as their Native Counterparts
Most of our recombinant toxin crystals were formed at very high protein concentrations. They were beautiful-looking under polarized light under the microscope, see
From the structural alignment of the solved structures with their native counterparts, such as rec-αBtx-HAP complex, rec-αCTX, rec-kBtx, rec-mambalgin, or with their most homologous native counterparts (such as rec-Hannalgesin and rec-MTα, whose crystal structure were not reported, known structure of αCTX and MT1, respectively, were used as the alignment counterpart), it is clear that our rTFPs are almost identical to their natural counterparts, except one or two amino acids at the N-terminal, which is a unique mark for their recombinant origin; as illustrated in
Three-fingered proteins' (rTFPs) are a large collection of proteins (peptides) with important functions and applications. Traditionally, such proteins were isolated from the venom of the snakes, with very few recombinantly obtained in the lab with in depth analysis and verification. Because of their scarcity and unique properties and applications, these proteins are very expensive (at the level of hundreds to thousands of US dollars per milligrams) and some are not commercially available. κBtx, for example, a unique α3β2 nicotinic acetylcholine receptor binder, is not commercially available (personal communications). Because TFPs usually contain 4 to 5 pairs of disulfide bonds, it is usually very hard to recombinantly express them, and those commercially available are mostly purified from snake venoms. Some researchers used chemical synthesis that successfully obtained these rTFPs, such as mambalgin-1 and mambalgin-2 (Diochot et al., 2012; Mourier et al., 2016; Pan et al., 2014; Salinas et al., 2021; Schroeder et al., 2014; Sun et al., 2018). However, due to the high cost in chemical synthesis and limited yields, these successful attempts did not change the overall scenario for production of TFPs.
With our pipeline, however, milligrams to hundreds of milligrams of rTFPs could be obtained in the lab. Through extensive biochemical assays and structural analysis, we were able to show our rTFPs are almost identical to their native counterparts. Considering the fact that several of our rTFPs reached milligrams to hundreds of milligrams on a single lab-scale production cycle, these rTFP could thus replace their natural counterparts, and the method worth to be exploited for production of other TFPs further, which could be of general interest in the field.
rec-αBtx (V31)
Refolding Result: good
A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This application is a national phase application claiming benefit of priority under 35 U.S.C. § 371 to Patent Convention Treaty (PCT) International Application serial number PCT/US2023/011168, filed Jan. 19, 2023, now pending, which claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/300,892, filed Jan. 19, 2022, now expired. The aforementioned application is applications are expressly incorporated herein by reference in their entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
This invention was made with government support under GM064642 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/011168 | 1/19/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63300892 | Jan 2022 | US |
