Preparation method and application of fatty acid transporter FATP transmembrane domain

Abstract

The invention discloses a preparation method and application of a fatty acid transporter FATP transmembrane domain. The method comprises the following steps: fusing a gene for coding an FATP transmembrane domain with a TrpLE tag sequence, and performing high-efficiency expression in escherichia coli to form an inclusion body. Thirdly, primarily purifying the fusion protein by using nickel column affinity chromatography, and specifically releasing a target transmembrane peptide fragment through cyanogen bromide chemical cleavage; and finally, carrying out fine purification by adopting one-step reversed-phase high-performance liquid chromatography to obtain a milligram-grade high-purity peptide fragment. The purified peptide fragment can be successfully reconstructed in a plurality of membrane simulation systems such as Fos-choline-14 micelles or DMPC / DHPC (Dimethyl Polycarbonate / Dimethyl Hexaphthyl Polycarbonate) lipid saucers and the like. According to the method, high-level expression of the FATP transmembrane domain in escherichia coli is achieved, the purification process is simple, convenient and efficient, the final product is high in purity, the yield can reach the milligram level, and the method is suitable for stable isotope labeling and meets the requirements of fine structure research such as nuclear magnetic resonance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing the transmembrane domain of a fatty acid transporter FATP and its application. Background Technology

[0002] Fatty acid transport protein 3 (FATP3), also known as very long-chain acyl-CoA synthetase 3 (ACSVL3), is encoded by the gene SLC27A3 and is one of the 27 members of the solute transporter family. FATP3 plays a dual role in lipid metabolism: (1) as an acyl-CoA synthetase, it catalyzes the acylation of long-chain and very long-chain fatty acids to generate acyl-CoA derivatives; (2) it promotes fatty acid uptake by enhancing cell membrane permeability to fatty acids (Pei Z, Fraisl P, Shi X, Gabrielson E, Forss-Petter S, Berger J, Watkins PA (2013) Very long-chain acyl-CoA synthetase 3: overexpression and growth dependence in lung cancer. PLoS One 8(7):e69392; Stahl A (2004) A current review of fatty acid transport proteins (SLC27). Pflugers Arch 447 (5):722-727.).

[0003] FATP3 is a membrane-associated protein with a hydrophobic transmembrane region in its primary structure. Early analyses of transmembrane hydrophobicity and structural predictions suggested that FATP3 might contain 1 to 4 transmembrane helical segments. However, due to the high binding of FATP3 to fatty acids, predicting its hydrophobic region proved difficult. Considering the high sequence homology between FATP1 and FATP3, and based on epitope tagging experiments with FATP1, the FATP3 membrane topology is inferred to be an N-terminus anchored transmembrane and a C-terminus facing inward. In addition to the main transmembrane helical structure, FATP3 may possess additional membrane-associated regions to help it localize to the endoplasmic reticulum or other membrane structures. To date, a high-resolution structure of FATP3 has not been reported, and its precise conformation remains to be experimentally verified.

[0004] Highly unsaturated omega-3 fatty acids, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are crucial for the development and function of nerve and retinal tissues. FATP3, due to its expression in barrier tissues, has been proposed as a potential mediator of omega-3 fatty acid transport. However, direct functional evidence is currently lacking to demonstrate that FATP3 specifically promotes the uptake of DHA or EPA; its role in omega-3 fatty acid transport is mostly inferred through tissue-specific expression patterns and indirect functional correlations.

[0005] Currently, there is a lack of research on its high-resolution structure and the role of transmembrane domains (TMDs) in fatty acid recognition. Existing methods for expressing and purifying membrane proteins often face problems such as low expression levels, aggregation, and high toxicity, and traditional membrane mimicry systems (such as micelles) are difficult to maintain the native conformation and function of proteins. Therefore, developing an efficient and reproducible method for preparing FATP3-TMDs and establishing a membrane reconstruction system suitable for its functional studies is of significant scientific value. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an efficient, reproducible, and structural biology-suitable method for preparing FATP-TMD.

[0007] Another object of the present invention is to provide FATP-TMD prepared by the method and its reconstruction method in a membrane simulation system that retains its natural conformation and function.

[0008] Another object of the present invention is to provide the application of the above-mentioned FATP-TMD complex in studying its specific recognition mechanism with polyunsaturated fatty acids (especially DHA), and in screening FATP functional regulators.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a fatty acid transporter protein FATP transmembrane domain (FATP-TMD), comprising the following steps: (1) Provide a recombinant expression vector containing a sequence encoding FATP-TMD; (2) The recombinant expression vector was introduced into host cells to express and obtain the TrpLE-FATP-TMD fusion protein; (3) Lyse host cells, isolate and purify the TrpLE-FATP-TMD fusion protein; (4) The TrpLE-FATP-TMD fusion protein was cleaved with cyanide to release FATP-TMD; (5) Purify the cleaved product to obtain high-purity FATP-TMD.

[0010] Preferably, in step (1), the sequence encoding FATP-TMD is the sequence encoding FATP3-TMD, which encodes the amino acid sequence of human FATP3 from position 1 to 32, MAALLLLPLLLLPLLLLKLHLWPQLRWLPAD or a functionally equivalent fragment thereof. The amino acid sequence of human FATP3 from position 1 to 32 is shown in SEQ ID No. 1, and the UniProt ID is Q8N163.

[0011] Preferably, in step (1), the recombinant expression vector is the pMM-LR6 plasmid.

[0012] Preferably, in step (2), the host cell is Escherichia coli BL21(DE3) competent cells, and protein expression is induced at low temperature by isopropyl-β-D-thiogalactoside IPTG.

[0013] More preferably, in step (2), competent cells are cultured in LB medium containing 50 μg / mL kanamycin until OD. 600 The concentration was 0.6-0.7. After incubating on ice for 20 minutes, 0.2 mM IPTG was added and expression was induced at 20°C for 16 hours. Host cells were then collected by centrifugation.

[0014] More preferably, in step (2), for nuclear magnetic resonance studies, M9 medium containing 50 μg / mL kanamycin can be used instead of LB medium, and the 15N isotope-labeled protein is expressed in M9 medium, with 15NH4Cl (1 g / L) as the sole nitrogen source in the medium to produce the 15N isotope-labeled protein.

[0015] Preferably, in step (3), the TrpLE-FATP-TMD fusion protein is expressed in the form of inclusion bodies and purified by nickel column affinity chromatography.

[0016] More preferably, in step (3), the bacterial body is resuspended in lysis buffer (100 mM Tris-HCl, 200 mM NaCl, pH 8.0) and sonicated on ice; centrifuged (24,000×g, 30 min, 4℃) and the supernatant is discarded, while the inclusion body precipitate is retained; the precipitate is dissolved in a buffer containing 6 M guanidine hydrochloride, 50 mM Tris-HCl, 200 mM NaCl, 1% Triton X-100 (v / v), pH 8.0, and centrifuged again to collect the supernatant; the supernatant is incubated with Ni-NTA resin for 1 hour and column chromatography is performed. After packing the column, it is washed sequentially with 10 column volumes of 8 M urea and ultrapure water, and the fusion protein is eluted with 3 column volumes of 90% (v / v) formic acid, and then column volumes of ultrapure water are added.

[0017] More preferably, in step (4), solid bromine cyanide (CNBr) is added to the eluent obtained by the column chromatography to a final concentration of 0.2 g / mL, and the mixture is reacted in the dark under nitrogen atmosphere for 1 hour to cleave and release FATP-TMD.

[0018] Preferably, in step (5), purification is performed using reversed-phase high-performance liquid chromatography.

[0019] More preferably, in step (5), the above-mentioned FATP-TMD is transferred into a 2 kDa MWCO dialysis bag, dialyzed with ultrapure water, and then lyophilized; the lyophilized sample is dissolved in 50% (v / v) formic acid, filtered through a 0.45 μm filter membrane, and then subjected to gradient elution with a C3 reversed-phase column in an isopropanol / acetonitrile / water system containing 0.1% (v / v) trifluoroacetic acid (TFA), the target peak is collected, and the sample is lyophilized to obtain high-purity FATP-TMD.

[0020] Secondly, the present invention provides an FATP-TMD prepared by the above method.

[0021] Preferably, its molecular weight is 3580.91 Da (theoretical value 3580.59 Da) as determined by MALDI-TOF mass spectrometry.

[0022] Preferably, SDS-PAGE and nuclear magnetic resonance hydrogen-nitrogen heteronuclear single-quantum coherence spectroscopy (SMR) are used. 1 H- 15 N HSQC) proved that its purity was higher than 95% and that it had the correct α-helical fold structure.

[0023] Thirdly, the present invention provides a membrane-reconstructed FATP-TMD complex comprising the above-mentioned FATP-TMD and a membrane mimicry system; the ligand (DHA) is also dissolved in the membrane mimicry system.

[0024] Preferably, the membrane simulation system is a detergent micelle or a lipid bilayer disk; the lipid bilayer disk is composed of long-chain phospholipids and short-chain phospholipids.

[0025] More preferably, the detergent micelles are Fos-choline-14 micelles or DPC micelles.

[0026] More preferably, the lipid bilayer disk is a DMPC / DHPC bilayer disk or a POPC / DHPC bilayer disk; the long-chain phospholipid is selected from DMPC or POPC, the short-chain phospholipid is DH6PC, and the molar ratio (q value) of the long-chain to short-chain phospholipid is 0.4 to 0.5.

[0027] Fourthly, the present invention provides the application of the above-described membrane-reconstructed FATP-TMD complex in any of the following aspects: (1) Used to screen candidate compounds that can regulate FATP function; (2) Used to study the interaction between proteins and ligands; (3) As a sample for structural biology research, it is used to analyze the three-dimensional structure, dynamic properties and binding sites of the transmembrane domain by nuclear magnetic resonance spectroscopy.

[0028] Preferably, in application (2), the fatty acid is a polyunsaturated fatty acid.

[0029] More preferably, the polyunsaturated fatty acid is docosahexaenoic acid (DHA).

[0030] Fifthly, the present invention provides a method for identifying FATP transmembrane domain ligands, comprising the following steps: The candidate material was contacted with the membrane-reconstructed FATP-TMD complex described above; Whether chemical shift perturbation occurs in FATP-TMD is detected by nuclear magnetic resonance spectroscopy; If a chemical shift perturbation occurs, it indicates that the candidate substance is a ligand of the FATP transmembrane domain.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. High-efficiency preparation and high yield: By adopting the TrpLE fusion tag strategy, the problem of toxicity and low yield of hydrophobic transmembrane peptides expressed in E. coli was successfully solved, enabling FATP-TMD to accumulate at a high level in inclusion bodies, with a yield of milligrams per liter of culture, providing sufficient samples for subsequent structural biology research.

[0032] 2. High Purity and Structural Integrity: Highly homogeneous FATP-TMD was obtained through an optimized CNBr chemical cleavage and one-step RP-HPLC purification process. HSQC NMR spectroscopy revealed sharp peaks with well-dispersed chemical shifts in various membrane simulation environments, confirming its correct α-helical folding and good structural homogeneity. This meets the prerequisites for high-resolution NMR studies. This invention provides a reliable experimental material and a unique technical platform for elucidating the molecular mechanism of FATP-mediated fatty acid transport at the atomic level, and its application prospects are broad.

[0033] 3. Key Functional Discoveries: This invention demonstrates that the binding of FATP-TMD to DHA depends on the lipid bilayer environment. The FATP transmembrane domain can specifically recognize and bind ω-3 fatty acids (such as docosahexaenoic acid, DHA) in a lipid bilayer environment (e.g., amphoteric discs), but lacks this binding activity in a simple micelle environment. In the bilayer discs (q=0.4-0.5), DHA titration induces significant, dose-dependent chemical shift perturbations (CSP), indicating rapid exchange and specific binding; however, this phenomenon is not observed in conventional Fos-choline-14 micelles, even with the addition of excess DHA. This discovery reveals that the physicochemical properties provided by the lipid bilayer environment, such as lateral pressure and hydrophobic matching, are crucial for the formation of the functional conformation of FATP. Attached Figure Description

[0034] Figure 1 The graph shows the expression and purification of the FATP3-TMD domain (a: schematic diagram of FATP3-TMD expression and purification process; b: high performance liquid chromatography (HPLC) chromatogram of the cyanogen bromide cleavage mixture (detection wavelength 214 nm); c: SDS-PAGE analysis of FATP3-TMD, lane 1 is the cyanogen bromide cleavage product of the TrpLE-FATP3-TMD fusion protein, lane 2 is FATP3-TMD purified by reversed-phase HPLC; d: mass spectrum of FATP3-TMD).

[0035] Figure 2 Reconstruction of FATP3-TMD and DHA titration (a: FATP3-TMD reconstructed in Fos-choline-14) 1 H- 15 b: N HSQC spectrum; 1 H- 15 N HSQC spectrum; c: DHA titration series of FATP3-TMD reconstructed in Fos-choline-14; d: DHA titration series of FATP3-TMD reconstructed in DMPC / DH6PC.

[0036] Figure 3 FATP3-TMD in DPC micelles 1 H- 15 N HSQC spectrum (the NH resonance peak of the indole side chain of tryptophan residue appears in the low field region (approximately 10.2–10.55 ppm)).

[0037] Figure 4 For 1D 1Characteristic peaks of methyl groups in 1H NMR spectra (a: Fos-choline-14 micelles; b: bilayer micelles composed of DMPC / DH6PC; c: bilayer micelles composed of POPC / DH6PC).

[0038] Figure 5 FATP3-TMD in bilayer micelles composed of DMPC / DH6PC 1 H- 15 N HSQC spectra (a: q = 0.4; b: q = 0.5; c: superposition of q = 0.4 and q = 0.5 spectra; the NH resonance peak of the side chain indole of tryptophan residue appears in the low field region (approximately 10.1–10.3 ppm)).

[0039] Figure 6 FATP3-TMD in bilayer micelles composed of POPC / DH6PC 1 H- 15 N HSQC spectra (a: q = 0.4; b: q = 0.5; where q represents the molar ratio of lipid to detergent; the NH resonance peak of the side chain indole of tryptophan residue appears in the low field region (approximately 10.1–10.25 ppm)).

[0040] Figure 7 For chemical shift perturbation (CSP) analysis.

[0041] Figure 8 This is a flowchart of the preparation method for the fatty acid transport protein FATP transmembrane domain (FATP-TMD). Detailed Implementation

[0042] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. For process parameters not specifically specified, conventional techniques can be referred to. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0043] The materials required in this embodiment are as follows: Escherichia coli BL21(DE3) competent cells were purchased from Beijing Bomeide Gene Technology Co., Ltd. The pMM-LR6 vector was kindly provided by Professor Blacklow SC of Harvard Medical School. Kanamycin and trifluoroacetic acid (TFA) were purchased from Shanghai Maclean Biotechnology Co., Ltd. Isopropyl-β-D-thiogalactoside (IPTG) was supplied by BioFroxx. Ni-NTA resin (L00666) was purchased from Genscript Biotech Co., Ltd. Cyanogen bromide was purchased from Shanghai Aladdin Biotechnology Co., Ltd. Chromatographic grade acetonitrile and isopropanol were purchased from Beijing Merida Technology Co., Ltd. 15NH4Cl and D2O were purchased from the Cambridge Isotope Laboratory. Phospholipids 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycerol-3-phosphocholine (DHPC) were supplied by AvantiPolar Lipids, and Fos-choline-14 (Fos-14) was provided by Anatrace. Unless otherwise specified, all other reagents were domestically produced analytical grade.

[0044] Figure 8 The procedure for preparing the fatty acid transporter FATP transmembrane domain (FATP-TMD) is shown.

[0045] Example 1: Construction of FATP3-TMD recombinant plasmid A professional gene synthesis company was commissioned to synthesize a DNA fragment encoding amino acids 1-32 of the N-terminus of human FATP3 (UniProt Q8N163) (MAALLLLPLLLLPLLLLKLHLWPQLRWLPAD, amino acid sequence as shown in SEQ ID No. 1, UniProt ID is Q8N163), based on the codon preference of E. coli. This fragment was then inserted into the appropriately enzyme-digested pMM-LR6 vector to construct the recombinant plasmid pMM-LR6-FATP3-TMD. DNA sequencing was used to verify the correctness of the inserted sequence and the accuracy of the reading frame.

[0046] Example 2: Expression, isotope labeling and purification of FATP3-TMD The expression and purification process diagram is shown below. Figure 1 As shown in a.

[0047] Express: The validated plasmid was heat-shocked and transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured overnight (12-16 hours) at 37°C with shaking at 220 rpm. The cells were then transferred to 1 L of fresh LB medium (or M9 basal medium for isotope labeling) at an initial OD600 of 0.1 and cultured at 37°C until the OD600 reached 0.6-0.7. The culture was incubated on ice for 20 minutes, and IPTG was added to a final concentration of 0.2 mM. Expression was then induced for 16 hours at 20°C and 220 rpm. The cells were collected by centrifugation at 4,000 × g for 30 minutes, and the cell pellet was flash-frozen in liquid nitrogen and stored at -80°C.

[0048] Purification and identification: a. Cell lysis and inclusion body extraction: 1 L of cell pellet was resuspended in 30 mL of lysis buffer (100 mM Tris-HCl, 200 mM NaCl, pH 8.0) and lysed using an ultrasonic cell disruptor (250 W, 1.5 seconds on, 1.5 seconds off, total 18 minutes) in an ice-water bath. The cells were then centrifuged at 24,000 × g for 30 minutes at 4 °C. The supernatant was carefully discarded, and the inclusion body pellet containing the fusion protein was retained.

[0049] b. Denaturation, Dissolution, and Preliminary Purification: Add 30 mL of denaturation buffer (6 M guanidine hydrochloride, 50 mM Tris-HCl, 200 mM NaCl, 1% Triton X-100 (v / v), pH 8.0) to the precipitate and vortex to dissolve completely. Centrifuge under the same conditions and collect the supernatant. Incubate the supernatant with 1 mL of pre-equilibrated Ni-NTA resin at room temperature for 1 hour.

[0050] c. Washing and elution: The resin-sample mixture was packed into the chromatography column, washed with 10 mL of 8 M urea solution, and then washed with 10 mL of ultrapure water. Finally, it was eluted with 3 mL of 90% (v / v) formic acid, and then 0.5 mL of ultrapure water was added. The eluent was collected.

[0051] d. CNBr cleavage: Add approximately 0.7 g of solid CNBr to the eluent, ensuring complete dissolution to a concentration of approximately 0.2 g / mL. In a fume hood, purge the reaction tube with nitrogen to replace the air, and react in the dark for 1 hour.

[0052] e. Dialysis and preliminary treatment: Transfer the reaction solution to a 2 kDa MWCO dialysis bag and dialyze twice with 2 L of ultrapure water, 1 hour each time. Transfer the dialysate to centrifuge tubes and freeze-dry.

[0053] f. RP-HPLC purification: The lyophilized sample was dissolved in 1 mL of 50% (v / v) formic acid and filtered through a 0.45 μm microporous membrane. Purification was performed using an HPLC system equipped with a C3 reversed-phase column. Mobile phase A: water / acetonitrile (75:25 v / v, containing 0.1% (v / v) TFA); Mobile phase B: isopropanol / acetonitrile (75:25 v / v, containing 0.1% (v / v) TFA). Gradient elution was used: 0–5 min, 0% B; 5–65 min, 0% B to 100% B; 65–80 min, 100% B, flow rate 3 mL / min, detection wavelength 280 / 214 nm. The fraction corresponding to the main peak was collected (retention time approximately 39–41 min). Figure 1 b), after combining and freeze-drying, a white flocculent solid was obtained, which is high-purity FATP3-TMD. SDS-PAGE ( Figure 1c) The identity of the target product has been preliminarily confirmed.

[0054] g. Mass spectrometry identification: A small amount of purified sample was mixed with an equal volume of saturated α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution, spotted onto a MALDI target plate, dried, and detected using a MALDI-TOF mass spectrometer (Shimadzu MALDI-8020) in positive ion, linear mode. The measured molecular weight was 3580.91 Da, consistent with the theoretical value (3580.59 Da), confirming the product's accuracy. Figure 1 d).

[0055] Example 3: Membrane Remodeling of FATP3-TMD Micellar Reconstruction: Accurately weigh 15 mg of Fos-Choline-14 (or DPC) and lyophilized FATP3-TMD, and dissolve them together in 1 mL of NMR buffer (25 mM MES, pH 6.7) containing 6 M guanidine hydrochloride. Gently mix until completely dissolved. Transfer the solution to a 2 kDa MWCO dialysis cartridge (Thermo Scientific) and dialyze statically against 1 L of NMR buffer at 4°C for 14 hours. Replace with fresh NMR buffer and continue dialyzing at 4°C and 400 rpm for 4.5 hours with stirring. Concentrate the dialysate to a final volume of approximately 300 μL using a 10 kDa MWCO ultrafiltration centrifuge tube at 1,000 × g. Add 20 μL of D2O (for NMR lock field), mix well, and transfer to a matching Shigemi tube.

[0056] Figure 4 a is Fos-choline-14 micelles in 1D 1 The characteristic peak of methyl groups in the 1H NMR spectrum indicates that the sample still contains Fos-choline-14 after dialysis, and the protein is reconstituted in micelles.

[0057] Figure 2 a is the NMR spectrum of FATP3-TMD reconstructed in Fos-choline-14 micelles; Figure 3 The NMR spectrum of FATP3-TMD reconstructed in DPC micelles was obtained; Fos-choline-14, which has a NMR signal number closer to the theoretical expectation, a sharper peak shape, and higher resolution, was selected for NMR structural characterization and DHA binding function verification.

[0058] Fat disc reconstruction: a. Take 10 mg DMPC (or POPC), 20 mg DH6PC and lyophilized FATP3-TMD dissolved in chloroform and dissolve them together in 1.5 mL hexafluoroisopropanol (HFIP).

[0059] b. In a fume hood, purge the solution with a gentle stream of nitrogen to form a uniform lipid-protein mixture film on the glass tube wall.

[0060] c. Transfer the glass tube to a freeze dryer and continuously evacuate for 12 hours to completely remove any residual organic solvents.

[0061] d. Add 500 μL of NMR buffer containing 6 M guanidine hydrochloride to the dried membrane and vortex to aid dissolution.

[0062] e. Transfer the dissolved mixture into a 2 kDa MWCO dialysis cartridge and dialyze against 500 mL of NMR buffer: allow to stand at room temperature for 30 minutes, then dialyze for 2.5 hours with stirring at 400 rpm. Replace with fresh NMR buffer and continue dialyzing at 400 rpm for 3 hours.

[0063] f. After dialysis, the sample should be clear and transparent. If slight turbidity occurs, a small amount of DH6PC can be added to observe whether the precipitation decreases. Finally, perform a brief low-speed centrifugation (e.g., 14,000 rpm, 5 min) and collect the supernatant.

[0064] g. Concentrate the clarified sample to approximately 300 μL, add 20 μL of D2O, mix well, and transfer to a Shigemi tube.

[0065] hq value verification and adjustment (q represents the molar ratio of long-chain lipids to short-chain detergents): Take a small amount of sample and collect one-dimensional... 1 1H NMR spectrum. Characteristic peaks of DMPC (or POPC) (methyl) and DH6PC (methyl) were obtained by integrating them. Figure 4 b represents a bilayer micelle composed of DMPC / DH6PC in 1D 1 Characteristic peak of methyl groups in 1H NMR spectrum; Figure 4 c represents a bilayer micelle composed of POPC / DH6PC in 1D. 1 (Methyl characteristic peak in the 1H NMR spectrum), calculate the actual molar ratio q = [DMPC] / [DH6PC]. If the q value deviates from the target value (e.g., 0.5), it can be fine-tuned by adding an appropriate amount of DH6PC or by diluting and concentrating again, and then verified again by NMR.

[0066] The q-value is the ratio of long-chain lipids to short-chain detergents. If q is too large, the bicelle radius increases, rotation slows down, and the signal becomes broadened and weakened. If q is too small, there is too much detergent, and the system almost becomes micelles, losing the bilayer properties of the membrane and hindering the native conformation of membrane proteins. Therefore, a suitable q-value needs to be selected based on the HSQC spectrum to ensure a strong NMR signal while maintaining bilayer properties, providing a near-native environment for membrane proteins.

[0067] Figure 5 Images a, 5b, and 5c show the NMR spectra of FATP3-TMD reconstruction in bilayer micelles composed of DMPC / DH6PC at q values ​​of 0.4 and 0.5. Figure 6 a and 6b show the NMR spectra of FATP3-TMD reconstruction in bilayer micelles composed of POPC / DH6PC at q values ​​of 0.4 and 0.5. Figure 5 NMR spectra of FATP3-TMD reconstructed in a bilayer micelle composed of DMPC / DH6PC; Figure 6 The NMR spectrum of FATP3-TMD reconstructed in a bilayer micelle composed of POPC / DH6PC was obtained; DMPC, which has a good peak shape and a broad chemical shift distribution, was selected for NMR structural characterization and DHA binding function verification.

[0068] Example 4: NMR structural characterization and DHA binding function verification Structural characterization: The sample prepared in Example 3 was placed in a Bruker Avance 800 MHz nuclear magnetic resonance spectrometer (equipped with a cryogenic probe) and FATP3-TMD was collected in Fos-choline-14 micelles and DMPC / DH6PC bilayer disks (q=0.5) at a constant temperature of 35°C. 1 H- 15 N HSQC spectrum. Spectral parameters: 1 H dimension has 2048 complex points. 15 N-dimensional, 160 complex points.

[0069] The results are as follows Figure 2 As shown in a and 2b, the indole NH resonance peak of the tryptophan residue side chain appears in the low-field region (approximately 10.1-10.5 ppm). Spectra with well-dispersed chemical shifts were obtained in both environments, proving that FATP3-TMD correctly folds into an α-helical structure. In particular, the spectrum in the bilayer disk shows more resolvable peaks (25) and sharper line shapes, indicating higher structural uniformity and stability in this environment.

[0070] DHA titration experiment: a. Prepare a 25 mM DHA NMR buffer stock solution (DHA dissolved in empty micelles or lipid discs).

[0071] b. Take 300 μL of FATP3-TMD sample reconstructed from a bilayer disk with a concentration of ~0.3 mM as the initial point (0 equivalent).

[0072] c. Using a microsyringe, gradually add specific volumes of DHA stock solution to the NMR tube, successively achieving a DHA:protein molar ratio of 0.5:1, 1:1, 2:1, 4:1, and 16:1. After each addition, gently mix the sample and collect data under these conditions. 1 H- 15 N HSQC spectrum.

[0073] d. As a control, the exact same titration procedure was performed on the FATP3-TMD sample reconstructed by Fos-choline-14 micelles.

[0074] Results and Analysis: a. such as Figure 2 As shown in d, the superimposed HSQC spectra show that the molar ratios of DHA to protein are 0:1 (red), 0.5:1 (blue), 1:1 (green), 2:1 (purple), 4:1 (pink), and 16:1 (gold), respectively. In the bilayer disk environment, with the addition of DHA, the peaks of multiple amino acid residues in FATP3-TMD showed continuous and progressive chemical shift perturbations (CSPs), and the degree of perturbation tended to saturate with increasing DHA concentration, which is a characteristic of a typical rapid exchange binding process.

[0075] b. On the contrary, such as Figure 2 As shown in c, the superimposed HSQC spectra show different molar ratios of DHA to protein as 0:1 (red), 0.5:1 (blue), 1:1 (green), 2:1 (purple), 4:1 (pink), and 16:1 (gold). In the micelle environment, even when the DHA concentration reached 16 times, no significant changes were observed in the HSQC spectra, proving that functional binding could not be formed in this environment.

[0076] c. Quantitative analysis of CSP data in double-layer disks (results as follows) Figure 7 As shown in the figure, the key residues most sensitive to DHA binding can be identified, providing clear targets for subsequent site-directed mutagenesis and mechanism studies.

[0077] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects: 1. High-efficiency preparation and high yield: By adopting the TrpLE fusion tag strategy, the problem of toxicity and low yield of hydrophobic transmembrane peptides expressed in E. coli was successfully solved, enabling FATP-TMD to accumulate at a high level in inclusion bodies, with a yield of milligrams per liter of culture, providing sufficient samples for subsequent structural biology research.

[0078] 2. High Purity and Structural Integrity: Highly homogeneous FATP-TMD was obtained through an optimized CNBr chemical cleavage and one-step RP-HPLC purification process. HSQC NMR spectroscopy revealed sharp peaks with well-dispersed chemical shifts in various membrane simulation environments, confirming its correct α-helical folding and good structural homogeneity. This meets the prerequisites for high-resolution NMR studies. This invention provides a reliable experimental material and a unique technical platform for elucidating the molecular mechanism of FATP-mediated fatty acid transport at the atomic level, and its application prospects are broad.

[0079] 3. Key Functional Discoveries: This invention demonstrates that the binding of FATP-TMD to DHA depends on the lipid bilayer environment. The FATP transmembrane domain can specifically recognize and bind ω-3 fatty acids (such as docosahexaenoic acid, DHA) in a lipid bilayer environment (e.g., amphoteric discs), but lacks this binding activity in a simple micelle environment. In the bilayer discs (q=0.4-0.5), DHA titration induces significant, dose-dependent chemical shift perturbations (CSP), indicating rapid exchange and specific binding; however, this phenomenon is not observed in conventional Fos-choline-14 micelles, even with the addition of excess DHA. This discovery reveals that the physicochemical properties provided by the lipid bilayer environment, such as lateral pressure and hydrophobic matching, are crucial for the formation of the functional conformation of FATP.

Claims

1. A method for preparing a fatty acid transporter FATP transmembrane domain (FATP-TMD), characterized in that, Includes the following steps: (1) Provide a recombinant expression vector containing a sequence encoding FATP-TMD; (2) The recombinant expression vector was introduced into a host cell to express the TrpLE-FATP-TMD fusion protein; (3) Lyse host cells, isolate and purify the TrpLE-FATP-TMD fusion protein; (4) The TrpLE-FATP-TMD fusion protein was cleaved with bromide cyanide to release FATP-TMD; (5) Purify the cleaved product to obtain high-purity FATP-TMD.

2. The preparation method according to claim 1, characterized in that, In step (1), the sequence encoding FATP-TMD is the sequence encoding FATP3-TMD, which encodes the amino acid sequence MAALLLLPLLLLPLLLLKLHLWPQLRWLPAD or its functional equivalent fragment of human FATP3. The amino acid sequence of human FATP3 from position 1 to position 32 is shown in SEQ ID No.1, and the UniProt ID is Q8N163.

3. The preparation method according to claim 1, characterized in that, In step (1), the recombinant expression vector is pMM-LR6 plasmid; in step (2), the host cell is Escherichia coli BL21(DE3) competent cells, and protein expression is induced at low temperature by isopropyl-β-D-thiogalactoside IPTG; in step (3), the TrpLE-FATP-TMD fusion protein is expressed in the form of inclusion bodies and purified by nickel column affinity chromatography; in step (5), the purification is performed by reversed-phase high-performance liquid chromatography.

4. An FATP transmembrane domain FATP-TMD prepared by the method of any one of claims 1-3.

5. A membrane-reconstructed FATP-TMD complex, characterized in that, It includes the FATP-TMD and membrane simulation system as described in claim 4.

6. The membrane-reconstructed FATP-TMD complex according to claim 5, characterized in that, The membrane simulation system is a detergent micelle or a lipid bilayer disk; wherein the lipid bilayer disk is composed of long-chain phospholipids and short-chain phospholipids.

7. The membrane-reconstructed FATP-TMD complex according to claim 6, characterized in that, The detergent micelles are Fos-choline-14 micelles or DPC micelles; the lipid bilayer disks are DMPC / DHPC bilayer disks or POPC / DHPC bilayer disks; the long-chain phospholipids are selected from DMPC or POPC, the short-chain phospholipids are DH6PC, and the molar ratio of long-chain to short-chain phospholipids q = 0.4~0.

5.

8. The use of the FATP-TMD of claim 4 or the membrane-reconstructed FATP-TMD complex of any one of claims 5-7 in any of the following aspects: (1) Used to screen candidate compounds that can regulate FATP function; (2) Used to study the interaction mechanism between proteins and ligands; (3) As samples for structural biology research, especially for nuclear magnetic resonance spectroscopy analysis.

9. The application according to claim 8, characterized in that, In application (2), the fatty acid is a polyunsaturated fatty acid.

10. A method for identifying FATP transmembrane domain ligands, characterized in that, Includes the following steps: The candidate material is contacted with the membrane-reconstructed FATP-TMD complex according to any one of claims 5-7; Whether the FATP-TMD has undergone chemical shift perturbation was detected by nuclear magnetic resonance spectroscopy; If a chemical shift perturbation occurs, it indicates that the candidate substance is a ligand of the FATP transmembrane domain.

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