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Jun 2019
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Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease
利用OaAEP1和TEV蛋白酶进行蛋白聚合物/多聚蛋白的酶促构建   

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Abstract

The development of chemical and biological coupling technologies in recent years has made possible of protein polymers engineering. We have developed an enzymatic method for building polyproteins using a protein ligase OaAEP1 (asparagine endopeptidase 1) and protease TEV (tobacco etching virus). Using a mobile TEV protease site compatible with the OaAEP1 ligation, we achieved a stepwise polymerization of the protein on the surface. The produced polyprotein can be verified by protein unfolding scenario using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides an alternative method for polyprotein engineering and immobilization.

Keywords: Polymerization (聚合), Enzyme (酶), Single-molecule force spectroscopy (单分子力谱), Polyprotein engineering (多聚蛋白质工程), OaAEP1 (OaAEP1)

Background

Several methods based on biochemical reactions for protein polymerization have been developed. For example, one approach is to design protein monomer with additional cysteines as the basic unit for ligation (Dietz et al., 2006; Durner et al., 2017). Another approach is to build the complete gene for the artificial protein oligomer (Carrion-Vazquez et al., 1999; Giganti et al., 2018). However, the engineering of large-sized protein polymer is often challenging. To address this challenge, we have developed an enzyme-mediated method that builds protein polymers/polyproteins in a stepwise fashion, using protein ligase and protease (Yang et al., 2017; Deng et al., 2019). When a TEV site (ENLYFQ/G) plus a leucine (L) is designed as ENLYFQ/GL-POI (POI: Protein of Interest) at the N-terminus of the protein unit, TEV cleavage produces an N-terminal GL residue of GL-POI, which is compatible with further OaAEP1 ligation. Our enzymatic method provides a new method for the preparation of the polyprotein sample with a controlled sequence and suitable for single-molecule studies, especially for complex protein systems.

The engineered polyprotein sequence is confirmed by individual protein unfolding event using AFM-SMFS. Besides a powerful imaging tool (Mannix et al., 2018), AFM can manipulate single molecule mechanically and directly measure its unfolding, unbinding, and rupture force (He et al., 2012; Scholl and Marszalek, 2018; Zhang et al., 2019). Thus, it is widely used to study protein (un)folding and mechanics (Infante et al., 2019; Krieg et al., 2019), protein-protein/ligand-receptor interaction (Ott et al., 2017; Zhang et al., 2019) and even chemical bond (Pill et al., 2019; Song et al., 2019; Yuan et al., 2019). Together with other established polyprotein engineering and immobilization methods (Dietz et al., 2006; Popa et al., 2013; Hoffmann et al., 2015; Walder et al., 2017), our enzymatic methodology can improve the quality and efficiency of SMFS study.

Materials and Reagents

  1. Glass coverslip (Sail Brand, China)
  2. E. coli BL21(DE3) (-80 °C)
  3. pQE80L-POI or pET28a-POI plasmid (Vector information in the Supplemetal file)
  4. Milli-Q water (18.2 MΩ/cm)
  5. Luria-Bertani (LB) medium (BD Difco)
  6. Iron (III) chloride hexahydrate (99%, Energy chemical)
  7. Calcium chloride hydrate crystalline aggregate (99.9965%, Alfa Aesar)
  8. (3-aminopropyl) triethoxysilane (APTES, 99%, Sigma-Aldrich)
  9. Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC, Thermo Scientific)
  10. Sodium chloride (NaCl) (99%, BBL Life Science)
  11. Tris(hydroxymethyl) aminomethane (Tris, > 99.9%, Sangon Biotech)
  12. EDTA (99.5%, Macklin)
  13. 5,5'-Dithiobis (2-nitrobenzoic Acid) (99%, Alfa Aesar)
  14. Ampicillin sodium salt (99% Zhu YanBIO)
  15. Kanamycin (≥ 750 μg/mg, Diamond)
  16. Magnesium sulfate (MgSO4) (AR, Shanghai Lingfeng Chemical Reagent Co., Ltd)
  17. Calcium chloride (CaCl2) (AR, Aladdin)
  18. D(+)-Glucose (AR, Aladdin)
  19. IPTG (Isopropyl-β-d-thiogalactopyranoside) (99% Zhu YanBIO)
  20. DNase (from Bovine Pancreas, Activity ≥ 500 Kunitz U/mg, Sangon Biotech)
  21. RNase (from Bovine Pancreas, Activity ≥ 60 U/mg, Sangon Biotech)
  22. PMSF (Zhu YanBIO)
  23. Potassium chromate (99.5%, Macklin)
  24. Concentrated sulfuric acid (AR, Sinopharm Chemical Reagent Co.,Ltd)
  25. Ethyl alcohol (AR, Sinopharm Chemical Reagent Co.,Ltd)
  26. Imidazole (99% Aladdin)
  27. Urea (AR, Sinopharm Chemical Reagent Co.,Ltd)
  28. TEV protease (produced by ourselves)
  29. OaAEP1 (produced by ourselves)
  30. BamHI (TaKaRa)
  31. BglII (TaKaRa)
  32. KpnI (TaKaRa)
  33. T4 Ligase (TaKaRa)
  34. Glycerol (99%, Macklin)
  35. M9 medium (see Recipes)
  36. Chromic acid (see Recipes)
  37. Lysis buffer (see Recipes)
  38. Wash buffer (see Recipes)
  39. Elution buffer (see Recipes)
  40. DTNB solution (see Recipes)
  41. TEV protease solution (see Recipes)
  42. AFM measurement buffer (see Recipes)

Equipment

  1. Avanti JXN-30 Centrifuge (Beckman Coulter)
  2. AKTA FPLC system (GE Healthcare)
  3. Mono Q 5/50 GL (GE Healthcare)
  4. NanoDrop 2000 spectrophotometer (Thermo Scientific)
  5. Nanowizard 4 AFM (JPK Instruments AG)
  6. Silicon nitride cantilever (MLCT, Bruker Corp)
  7. Sonictor (Biosafer 650-92)

Software

  1. JPK data processing (developed by JPK Instruments AG)
  2. Igor Pro 6.12 (Wavemetrics)

Procedure

  1. Gene cloning
    1. Use three-restriction digestion enzyme system BamHI-POI-BglII-stop codon-KpnI for connecting the gene of different protein fragments. In this enzyme system use BglII and KpnI digest to produce vector and use BamHI and KpnI digest to produce the insert. As BamHI digestion and BglII digestion leads to the same cohesive end (GATC), it is possible to use T4 ligase connect the vector and the insert.
    2. Confirm all genes by sequencing.

  2. Expression and production of interest proteins
    Note: In the experimental operation, always pay attention to the aseptic operation. Different proteins have different E. coli harvest shelf life. It is necessary to test the shelf life for the specific protein.
    1. Transform the pQE80L-POI or pET28a-POI plasmid into E. coli BL21(DE3) cell.
    2. Apply the bacterial solution to the LB plate containing ampicillin sodium salt (100 μg/ml) for pQE80L plasmid or kanamycin (50 μg/ml) for pET28a plasmid and incubate at 37 °C for 14-16 h.
    3. Pick single colonies into 15 ml LB medium containing ampicillin sodium salt (100 μg/ml) or kanamycin (50 μg/ml). Keep shaking at 200 rpm at 37 °C for 16-20 h.
    4. After grown saturation, dilute the overnight cultures into 800 ml fresh LB media (add to the LB medium in a ratio of 1:50) containing respective antibiotics (the concentration of antibiotics is the same as above). As for the metalloprotein, centrifuged the culture at 1,800 x g and resuspended the precipitation by 16 ml M9 medium, and then add to 800 ml respective antibiotics containing M9 medium as well.
    5. Shake culture at 200 rpm at 37 °C until the optical density at 600 nm (OD600) reaches 0.6-0.8 (this takes about 2.5-3 h, while M9 medium always takes about 5-6 h). Reserve a 100-μl sample of the culture as the pre-induction control for testing protein expression.
    6. Add IPTG (Isopropyl-β-d-thiogalactopyranoside) to a final concentration of 1 mM to induce protein expression, while metalloprotein always needs some additional metal ion, and shake the culture at 200 rpm at 37 °C for 4-6 h.
    7. Harvest the culture at 13,000 x g for 25 min at 4 °C and store at -80 °C before purification.

  3. Purification of interest proteins
    1. Resuspend the cells in 25 ml lysis buffer (contained 0.06 mg DNase, 0.06 mg RNase, 0.08 mg PMSF) and produce the lysis using a Biosafer sonicator (15% amplitude) on ice for 30 min. Clarify the cell lysate at 19,600 x g for 40 min at 4 °C and collect the supernatant fraction.
    2. Pack a Poly-Prep column with 1-1.5 ml (bed volume) of Co-NTA or Ni-NTA affinity column and wash it with ten column volumes (CV) of Milli-Q water and then 10 CVs wash buffer (by gravity flow).
    3. Load the supernatant onto the column by gravity flow and collect the flow-through. Load the flow-through onto the column by gravity and collect the secondary flow-through. Load the secondary flow-through onto the column by gravity and finish the sample loading.
    4. Wash the column with 50 CVs of wash buffer.
    5. Elute the bound protein with 3 CVs of elution buffer at 4 °C for 15 min. Moreover, carry out anion exchange purification for pure metal form rubredoxin protein using AKTA with Mono Q column at pH 8.5 at 4 °C.
    6. Analyze the samples of the eluate and control group by SDS-PAGE.
    7. Use the Ellman method to confirm the concentration of ELPs, in which 10 μl protein incubated with 20 μl DTNB solution for 30 min at room temperature and the solution composited with 10 μl the elution buffer solution and 20 μl DTNB solution served as blank to detect the absorbance of protein and DTNB reaction solution at 412 nm (ε = 13,700 M−1 cm−1) by NanoDrop 2000 spectrophotometer.

  4. Functionalized coverslip surface preparation
    Note: The functionalized coverslip should be immediately used or stored at 4 °C for a month after preparation.
    1. Clean and activate glass coverslips by immersing in chromic acid at 80 °C for 30 min. Wash away chromic acid traces on the coverslips by water and then ethyl alcohol. Dry the coverslips by a stream of nitrogen.
    2. Aminosilylate the coverslips by immersing them in 1% (v/v) APTES toluene solution for 1 h at room temperature avoiding light.
    3. Wash the coverslips with toluene and absolute ethyl alcohol and dry the coverslips with nitrogen.
    4. Bake the coverslips at 80 °C for 15 min.
    5. Add 200 μl sulfo-SMCC (1 mg/ml) dimethyl sulfoxide (DMSO) solution between two immobilized coverslips after the coverslips cooling down to room temperature and incubate for 1 h protected from light at room temperature.
    6. Wash every piece of the coverslips with 15 ml DMSO first and then with absolute ethyl alcohol to remove residual sulfo-SMCC. Use a stream of nitrogen to dry the coverslips.
    7. Stick cleaned quartz ring on the functioned side of the coverslip to build a chamber.
    8. Pipet 200 μl of 200 μM GL-ELP50nm-C protein solution onto the functionalized coverslip in the chamber and incubate for approximately 3 h.
    9. Wash the chamber with Milli-Q water to remove the unreacted GL-ELP50nm-C.

  5. Functionalized cantilevers surface preparation
    Note: Silicon nitride cantilever was used as a force probe. The surface chemistry of the cantilevers was similar to that of the coverslip.
    1. Immerse the cantilevers at 80 °C for 10 min by chromic acid treatment. Remove the traces of chromic acid by water first and then ethyl alcohol. Use a piece of filter paper blot ethyl alcohol traces up.
    2. Functionalize the cleaned cantilevers by amino-silanization with APTES and then conjugate the cantilevers to sulfo-SMCC as the method mentioned in Section D, Functionalized coverslip surface preparation.
    3. Immerse the functionalized cantilever with Cys-ELP50nm-NGL at the concentration of 200 μM to the surface with maleimide group of sulfo-SMCC for 1.5 h.
    4. Wash the coverslip with Milli-Q water to remove the unreacted Cys-ELP50nm-NGL and store the cantilever at -20 °C.
    5. Before the AFM experiment, immerse a ELP-functionalized cantilever in 50 μM GL-CBM-XDoc protein solution with 200 nM OaAEP1 for 20-30 min at 25 °C, and then wash away the unreacted proteins by 15-20 ml AMF buffer.

  6. Stepwise polyprotein preparation with controlled sequences
    Note: Use 15-20 ml AFM buffer to wash away residual proteins after each reaction step. Figure 1 presents the schematic diagram of the controlled sequence polyprotein preparation.
    1. Mix unit Coh-tev-L-POI-NGL(50 μM) and OaAEP1 (200 nM) and add 60 μl to the functionaliazed coverslip. link the ligation unit Coh-tev-L-POI-NGL to the functionalized coverslip.
    2. Add 100 μl TEV protease solution to cleave the TEV site in the protein unit for about 1 h at 25  °C. And GL-Ub-NGL-glass will produce with exposed N-terminal GL residues.
    3. Repeat Steps F1-F2 (N-1) times to get the protein-polymer GL-(Ub)N-NGL-Glass. After OaAEP ligation of the final ligation unit, do not take the TEV cleavage reaction to obtain Coh-tev-L-(Ub)n-NGL-Glass.


      Figure 1. The schematic displays the stepwise ligation and cleavage procedure to produce sequence-controlled polyprotein on the glass surface and probed with an XDoc-protein maker-modified cantilever

  7. AFM measurements
    Note: The AFM experiment parameters (Z length, Z speed, setpoint, sample rate and duration time) are adjustable in different experiments.
    1. Add 1 ml AFM buffer containing10 mM CaCl2 to the chamber (Figure 2).


      Figure 2. A photo image about AFM chamber with 1 ml AFM buffer solution. The black ring shows the position of the polyprotein.

    2. Move the laser to the D tip of the functionalized probe (Figure 3). Use the equipartition theorem to calibrate the spring constant (κ) of each cantilever in solution with an accurate value before the experiment.


      Figure 3. CCD picture of the cantilever tip in the AFM experiment. The triangle which the red ring located is D tip. The red ring points out the lase radiation position on D tip.

    3. The cantilever tip attached to the sample surface and hold at this position for a while to formed the cohesin/dockerin pair as shown in Figure 4, segment 0 and segment 1.
    4. Retracted the cantilever from the surface at a constant velocity of 400 nm s−1 while recording the distance and cantilever deflection at a sampling rate of 4,096 Hz. As shown in Figure 4, segment 2.
    5. Hold the cantilever for a while to relax the cohesin/dockerin pair as shown in Figure 4, segment 3.


      Figure 4. The dialog about the AFM experiment parameter setting. One AFM measurement contain four step. Segment 0 makes cantilever move towards the sample surface. Segment 1 keep the cantilever staying at the height reached by segment 0 for duration time. Segment 2 retract the cantilever from the surface and the protein unfold during this period. Segment 3 keep the cantilever at the height reached by segment 2 for duration time.

Data analysis

  1. Use JPK data processing select force-extension traces.
  2. Use Igor Pro 6.12 (Wavemetrics) analysis the data traces. Only the data traces containing all the specific contour length increments belong to fingerprint protein domains [GB1(H18 nm) and CBM (H58 nm)] and a high rupture force from the cohesin-dockerin dissociation meet the requirement (more than 200-300 pN) (Figure 5). Fit The curves by the worm-like-chain (WLC) model of polymer elasticity with the persistence length of ~0.4 nm.
  3. Use Gauss model fit the histograms of unfolding forces to determine values of the most probable unfolding force () and contour length increment (<∆Lc>).


    Figure 5. Typical force versus distance trace for the sequence-controlled polyprotein, Coh-Ub4, containing signals of CBM unfolding, dockerin-cohesin pair rupture and four copies of Ub

Recipes

  1. Lysis buffer
    50 mM Tris
    150 mM NaCl
    pH 7.4
  2. Wash buffer
    20 mM Tris
    400 mM NaCl
    2 mM imidazole
    pH 7.4
  3. Elution buffer
    20 mM Tris
    400 mM NaCl
    250 mM imidazole
    pH 7.4
  4. DTNB solution
    0.5 mM 5,5'-Dithiobis (2-nitrobenzoic Acid)
    1 mM EDTA
    7.2 M Urea
  5. TEV protease solution
    0.4 mg/ml TEV protease
    75 mM NaCl
    0.5 mM EDTA
    25 mM Tris-HCl, pH 8.0
    10% [v/v] glycerol
  6. AFM measurement buffer
    100 mM Tris
    100 mM NaCl
    pH 7.4
  7. Chromic acid
    20 g potassium chromate
    40 ml ultrapure water
    360 ml concentrated sulfuric acid
  8. M9 medium
    0.4% glucose
    0.1 mM CaCl2
    2 mM MgSO4

Acknowledgments

National Natural Science Foundation of China (Grant Nos. 21771103, 21977047), Natural Science Foundation of Jiangsu Province (Grant No. BK20160639). This protocol was modified from previous work “Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level (Deng et al., 2019).

Competing interests

The authors declare no conflict of interest.

References

  1. Carrion-Vazquez, M., Oberhauser, A. F., Fowler, S. B., Marszalek, P. E., Broedel, S. E., Clarke, J. and Fernandez, J. M. (1999). Mechanical and chemical unfolding of a single protein: A comparison. Proc Natl Acad Sci U S A 96(7): 3694-3699. 
  2. Deng, Y., Wu, T., Wang, M., Shi, S., Yuan, G., Li, X., Chong, H., Wu, B. and Zheng, P. (2019). Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level. Nat Commun 10(1): 2775. 
  3. Dietz, H., Bertz, M., Schlierf, M., Berkemeier, F., Bornschlogl, T., Junker, J. P. and Rief, M. (2006). Cysteine engineering of polyproteins for single-molecule force spectroscopy. Nat Protoc 1(1): 80-84.
  4. Durner, E., Ott, W., Nash, M. A. and Gaub, H. E. (2017). Post-translational sortase-mediated attachment of high-strength force spectroscopy handles. Acs Omega 2(6): 3064-3069. 
  5. Giganti, D., Yan, K., Badilla, C. L., Fernandez, J. M. and Alegre-Cebollada, J. (2018). Disulfide isomerization reactions in titin immunoglobulin domains enable a mode of protein elasticity. Nat Commun 9(1): 185. 
  6. He, C., Genchev, G. Z., Lu, H. and Li, H. (2012). Mechanically untying a protein slipknot: multiple pathways revealed by force spectroscopy and steered molecular dynamics simulations. J Am Chem Soc 134(25): 10428-10435. 
  7. Hoffmann, T., Tych, K. M., Crosskey, T., Schiffrin, B., Brockwell, D. J. and Dougan, L. (2015). Rapid and robust polyprotein production facilitates single-molecule mechanical characterization of β-Barrel assembly machinery polypeptide transport associated domains. ACS Nano 9(9): 8811-8821.
  8. Infante, E., Stannard, A., Board, S. J., Rico-Lastres, P., Rostkova, E., Beedle, A. E. M., Lezamiz, A., Wang, Y. J., Gulaidi Breen, S., Panagaki, F., Sundar Rajan, V., Shanahan, C., Roca-Cusachs, P. and Garcia-Manyes, S. (2019). The mechanical stability of proteins regulates their translocation rate into the cell nucleus. Nat Phys 15: 973-981.
  9. Krieg, M., Fläschner, G., Alsteens, D., Gaub, B. M., Roos, W. H., Wuite, G. J. L., Gaub, H. E., Gerber, C., Dufrêne, Y. F. and Müller, D. J. (2019). Atomic force microscopy-based mechanobiology. Nat Rev Phys 1(1): 41-57. 
  10. Mannix, A. J., Zhang, Z. H., Guisinger, N. P., Yakobson, B. I. and Hersam, M. C. (2018). Borophene as a prototype for synthetic 2D materials development. Nat Nanotech 13(6): 444-450. 
  11. Ott, W., Jobst, M. A., Schoeler, C., Gaub, H. E. and Nash, M. A. (2017). Single-molecule force spectroscopy on polyproteins and receptor–ligand complexes: The current toolbox. J Stru Bio 197(1): 3-12. 
  12. Pill, M. F., East, A. L. L., Marx, D., Beyer, M. K. and Clausen-Schaumann, H. (2019). Mechanical activation drastically accelerates amide bond hydrolysis, matching enzyme activity. Angew Chem Int Ed 58(29): 9787-9790. 
  13. Popa, I., Berkovich, R., Alegre-Cebollada, J., Badilla, C. L., Rivas-Pardo, J. A., Taniguchi, Y., Kawakami, M. and Fernandez, J. M. (2013). Nanomechanics of HaloTag Tethers. J Am Chem Soc 135(34): 12762-12771. 
  14. Scholl, Z. N. and Marszalek, P. E. (2018). AFM-based single-molecule force spectroscopy of proteins. Methods Mol Biol 1814: 35-47. 
  15. Song, Y., Ma, Z., Yang, P., Zhang, X., Lyu, X., Jiang, K. and Zhang, W. (2019). Single-molecule force spectroscopy study on force-induced melting in polymer single crystals: the chain conformation matters. Macromolecules 52(3): 1327-1333. 
  16. Walder, R., LeBlanc, M. A., Van Patten, W. J., Edwards, D. T., Greenberg, J. A., Adhikari, A., Okoniewski, S. R., Sullan, R. M. A., Rabuka, D., Sousa, M. C. and Perkins, T. T. (2017). Rapid characterization of a mechanically labile α-helical protein enabled by efficient site-specific bioconjugation. J Am Chem Soc 139(29): 9867-9875. 
  17. Yang, R., Wong, Y. H., Nguyen, G. K. T., Tam, J. P., Lescar, J. and Wu, B. (2017). Engineering a catalytically efficient recombinant protein ligase. J Am Chem Soc 139(15): 5351-5358.
  18. Yuan, G., Liu, H., Ma, Q., Li, X., Nie, J., Zuo, J. and Zheng, P. (2019). Single-molecule force spectroscopy reveals that iron-ligand bonds modulate proteins in different modes. J Phys Chem Lett 10(18): 5428-5433. 
  19. Zhang, S., Qian, H.-j., Liu, Z., Ju, H., Lu, Z.-y., Zhang, H., Chi, L. and Cui, S. (2019). Towards unveiling the exact molecular structure of amorphous red phosphorus by single-molecule studies. Angew Chem Int Ed 58(6): 1659-1663.

简介

[摘要 ] 发展化学和生物耦合技术,近年来已成为可能中蛋白质聚合物工程。我们已经开发了酶法用于构建多蛋白使用蛋白连接酶OaAEP1(天冬酰胺内肽酶1)和蛋白酶TEV(烟草蚀刻病毒)。使用与OaAEP1连接兼容的移动TEV蛋白酶位点,我们实现了蛋白质在表面上的逐步聚合。产生的多蛋白可以使用基于原子力显微镜的单分子力谱(AFM-SMFS)通过蛋白质展开场景来验证。因此,这项研究为多蛋白工程和固定化提供了另一种方法。

[背景 ] 已经开发了几种基于生化反应的蛋白质聚合方法,例如,一种方法是设计以额外的半胱氨酸为连接基本单位的蛋白质单体(Dietz 等,2006 ; Durner 等,2017)。)。另一种方法是构建完整的基因的人工蛋白质寡聚物(腐肉巴斯克斯等人,1999年 ; Giganti 等人。,2018)。然而,该工程大尺寸高分子蛋白质往往是具挑战性这为了应对这一挑战,我们开发了一种酶介导的方法,可使用蛋白质连接酶和蛋白酶逐步构建蛋白质聚合物/多聚蛋白质(Yang 等人,2017 ; Deng 等人,2019)。 )在蛋白单元的N端加上一个亮氨酸(L)设计为ENLYFQ / GL-POI(POI:目的蛋白),TEV裂解产生GL-POI的N端GL残基,该残基可兼容进一步的OaAEP1连接。我们的合成方法提供了一种新方法 对于准备d的聚蛋白样品以受控的序列和适合单- 分子研究,特别是对于复杂的蛋白质系统。

通过使用AFM-SMFS的单个蛋白质解折叠事件可以确认工程化的多蛋白质序列。除了强大的成像工具(Mannix 等人,2018)之外,AFM还可以机械地操纵单分子并直接测量其解折叠,解链和断裂力(He 等等人,2012年;Scholl和Marszalek,2018年;Zhang 等人,2019年),因此,它被广泛用于研究蛋白质(非)折叠和力学(Infante 等人,2019年;Krieg 等人,2019年),蛋白质/蛋白质/配体-受体的相互作用(Ott 等人,2017 ; Zhang 等人,2019),甚至化学键(Pill 等人,2019 ; Song 等人,2019 ; Yuan 等人,2019)。与其他establis hed多蛋白工程和固定化方法一起使用(Dietz 等,2006 ; Popa 等,2013 ; Hoffmann 等,2015 ; Walder 等,2017),我们的理论方法可以提高蛋白质的质量和效率SMFS研究。

关键字:聚合, 酶, 单分子力谱, 多聚蛋白质工程, OaAEP1

材料和试剂


 


玻璃盖玻片(中国风帆品牌)
大肠杆菌 BL21(DE3)(-80°C)
pQE80L-POI或pET28a-POI质粒(Supplemetal文件中的矢量信息)。
Milli-Q水(18.2MΩ/ cm)
Luria-Bertani(LB)培养基(BD Difco )
六水合氯化铁(99%,能源化工)
Ç Alcium氯化物水合物结晶集合物(99.9965 Pasento,阿尔法Aesar公司)
(3- 一个Minopropyl)三乙氧基硅烷(APTES,99%,Sigma-Aldrich公司)
小号Ulfosuccinimidyl 4-(N- 马来酰亚胺甲基)环己烷-1-羧酸酯(磺基-SMCC,Thermo Scientific)进行
氯化钠(NaCl)(99 %,BBL生命科学)
Ť RIS(羟甲基)氨基甲烷至(Ť RIS ,> 99.9%,生工Biotech)的             
EDTA(99.5%,Macklin)
5,5'-二硫代双(2-硝基苯甲酸)(99%,Alfa Aesar )。
氨苄西林钠盐(99%朱艳生物)。
卡那霉素(≥ 750 MYU ģ /镁,金刚石)
硫酸镁(MgSO 4 )(AR,上海凌风化学试剂有限公司)
氯化钙(CaCl 2 )(AR,阿拉丁)
D(+)-葡萄糖(AR,阿拉丁)
IPTG(异丙基-β-d- 硫代吡喃半乳糖苷)(99%朱艳生物)
DNA酶(来自牛胰腺,活动≥ 500 Kunitz结构U /毫克,生工Biotech)的
RNase(牛胰腺中,活性≥60 U / mg,Sangon Biotech)
PMSF (朱彦生物)
铬酸钾(99.5%,Macklin)
浓硫酸(AR,国药化学试剂有限公司)
乙醇(AR,国药化学试剂有限公司)
咪唑(99%阿拉丁)
尿素(AR,国药化学试剂有限公司)
TEV蛋白酶(由我们自己生产)
OaAEP 1 (由我们自己生产)
的BamH 我(TaKaRa公司)
Bgl II (TaKaRa )
Kpn I (TaKaRa )
T4连接酶(TaKaRa )
甘油(99%,Macklin)
M9中号(请参阅食谱)
铬酸(请参阅食谱)
裂解缓冲液(请参见食谱)
洗涤缓冲液(请参见食谱)
洗脱缓冲液(请参见配方)
DTNB解决方案(请参阅食谱)
TEV蛋白酶溶液(请参阅食谱)
AFM测量缓冲区(请参阅配方)
 


配套设备


 


Avanti JXN-30离心机(贝克曼库尔特)
AKTA FPLC系统(GE Healthcare)
Mono Q 5/50 GL(GE Healthcare)
纳米d 罗普2000分光光度计(Thermo Scientific的)
Nanowizard 4 AFM(JPK Instruments AG)
氮化硅悬臂(MLCT,Bruker Corp)
声波发生器(Biosafer 650-92)
 


软体类


 


JPK数据处理(由JPK Instruments AG开发)
Igor Pro 6.12(波制)
 


程序


 


基因克隆
三限制使用消化酶系统的BamH 我-POI - 的Bgl II -Stop密码子KPN 我。连接不同蛋白片段的基因这种酶系统使用的Bgl II 和KPN 我文摘要生产向量和使用的BamH 我和KPN 我为了产生文摘的插入。作为的BamH 我消化的Bgl II 消化导致相同的粘性末端(GATC) ,它可以使用T4连接酶连接载体和插入。
通过测序确认所有基因。
 


目的蛋白的表达和产生
注意:在实验操作中,要始终注意无菌操作,不同的蛋白质在大肠杆菌中的保存期限不同,有必要测试特定蛋白质的保存期限。


将pQE80L-POI或pET28a-POI质粒转化到大肠杆菌BL21(DE3)细胞中。
将细菌溶液应用于含有pQE80L质粒的氨苄青霉素钠盐(100μg / ml )或用于pET28a质粒的卡那霉素(50μg / ml )的LB平板中,并在37°C下孵育14-16 h。
单菌落成拾15米升LB培养基含有氨苄青霉素钠盐(100 微克/米升)或卡那霉素(50 微克/米升),保持在200rpm在37℃下振荡℃下16-20小时。
生长饱和度,稀释后的过夜培养进入800米大号新鲜LB培养基(添加到LB培养基的比例为1 :50)包含。各抗生素(抗生素的浓度同上)作为金属蛋白,离心文化在1,8 00 ×g的和再悬浮沉淀- 16进制大号M9培养基中,然后加入800种大号含M9介质好各抗生素。
在37°C下以200 rpm的速度摇动培养物,直到600 nm(OD 600 )的光密度达到0.6-0.8(这大约需要2.5- 3 h,而M9 培养基总是需要大约5-6 h)。保留100- MYU 大号样的文化作为预感应控制对于检测蛋白表达。
加入IPTG(异丙基-β-d- 硫代巯基乳糖吡喃糖苷)至终浓度为1 mM以诱导蛋白质表达,而金属蛋白质始终需要一些额外的金属离子,并在37℃下以200 rp m 摇动培养物4-6 h 。
在4°C下以13 000 x g的浓度收获培养物25分钟,并在纯化前在-80°C下储存。
 


感兴趣蛋白的纯化
将细胞重悬在25男大号裂解缓冲液(所含0.06的Mg DNA酶,0.06 mg RNase,0.08 mg PMSF)并使用Biosafer 超声仪(15%振幅)在冰上裂解30分钟。以19,6 00 x g f 澄清细胞裂解液或在4 °C澄清40分钟并收集前体级分。
聚包准备柱1-1.5米大号(床体积)有限公司-NTA或Ni-NTA亲和柱,洗它十柱体积(CV)中的Milli-Q水,然后10个人简历洗涤液(通过重力流量)。
通过重力流将流加载到色谱柱上并收集流路;通过重力将流路加载到色谱柱上并收集二级流路; 通过重力将次级流路加载到色谱柱上并完成样品加载。
用50 CV 的洗涤缓冲液洗涤色谱柱。
用3 CV 的洗脱缓冲液在4°C下洗脱结合的蛋白质15分钟,此外,在4°C下使用带有Mono Q柱的AKTA用AKTA 对纯金属形式的氧化还原蛋白进行阴离子交换纯化。
通过SDS-PAGE 分析洗脱液和对照组的样品。
使用埃尔曼方法来确认电子学习产品的浓度,在这里10 MYU 大号蛋白孵育20 MYU 大号DTNB溶液中30分钟,在室温与解决方案合成随着10 MYU 大号洗脱缓冲液中,20 MYU 大号DTNB解决方案担任为了检测空白吸光度蛋白及DTNB反应溶液在412nm处(Ipushiron = 13700中中号-1 厘米-1 )通过纳米d 罗普2000分光光度计。  
 


盖玻片表面Preparatio官能Ñ
注意:功能化的盖玻片应立即使用,或在制备后于4°C下保存一个月。


在80°C的铬酸中浸泡30分钟,清洁并活化玻璃盖玻片,先用水,然后用乙醇冲洗盖玻片上的铬酸痕迹,再用氮气流干燥盖玻片。
将盖玻片在室温下浸入1%(v / v)APTES甲苯溶液中1小时,以使其氨基化,避免光照。
用甲苯和无水乙醇清洗盖玻片,并用氮气干燥盖玻片。
在80°C下烘烤盖玻片15分钟。
200添加MYU 大号磺基-SMCC(1毫克/米大号)之间的两个固定化盖玻片二甲基亚砜(DMSO)溶液压脚提升盖玻片在冷却至室温,并孵育1个小时避光在室温下。
清洗每一件COV来Erslips凭借15米大号d MSO 首先,然后用无水乙醇以去除残留的硫代-SMCC。使用流的氮干盖玻片小号。
将清洁的石英环粘在盖玻片的功能面上,以形成一个腔室。
200吸取  MYU 大号200  MyuM GL-ELP 50NM -C蛋白溶液涂布到官能盖玻片在腔室中并孵育约3 H.
用Milli-Q水清洗反应室,以除去未反应的GL-ELP 50nm -C。
 


功能化悬臂表面处理
注意:氮化硅悬臂用作力探针,悬臂的表面化学性质与盖玻片相似。


通过铬酸处理将悬臂在80°C的温度下浸泡10分钟,先用水先去除痕量铬酸,然后再去除乙醇,然后用滤纸吸干痕量乙醇。
清洁后悬臂功能化通过氨基- 硅烷化随着APTES和共轭然后悬臂要硫代-SMCC物的方法,提到在小号挠度d,官能盖玻片苏Rface准备。             
所述沉浸官能悬臂用Cys-ELP 50NM -NGL在200的浓度  MyuM 的表面处理,将马来酰亚胺基团的Sulfo -SMCC 1.5 H.
用Milli-Q水洗涤盖玻片以去除未反应的Cys-ELP 50nm -NGL,并将悬臂存储在-20°C。
在AFM实验之前,浸入甲ELP- 官能d 悬臂在50  MyuM GL-CBM- XDOC 蛋白质溶液和200  NM OaAEP1 对于20-30分钟,在25° C,再洗去未反应的蛋白质15-20中号大号AMF缓冲区。
 


具有受控序列的逐步多蛋白制备
注:使用15-20中号大号。AFM缓冲洗去残留的蛋白质每个反应步骤图1后给出了示意图的控制顺序多聚准备。


单位COH混合TEV-L-POI-NGL(50 MyuM)和OaAEP1(200纳米),加入60 MYU 大号到Functionaliazed盖玻片,链接结扎股COH-TEV-L-POI-NGL到官能盖玻片。
100添加  MYU 大号TEV蛋白酶溶液劈裂的TEV网站蛋白质组约1小时,在25   ° C. 而GL - 泛-NGL,玻璃将产生暴露的N终端GL残留。
重复步骤F1-F2 (N-1 )遍才能得到蛋白质-聚合物GL-(泛)ñ -NGL-玻璃后。OaAEP 结扎最后的结扎股,别拿TEV裂解反应,得到COH - Tev来- L-(Ub )n -NGL玻璃。
 


D:\重新格式化\ 2020-2-7 \ 1902794--1332彭正802285 \图jpg \图1.jpg


1.原理图图显示了阶梯式结扎和裂解步骤以产生序列控制下的多聚在玻璃表面和探索Sun Yatsen随着一个XDOC -蛋白Maker- 改性悬臂


 


AFM测量
注:牛逼他AFM实验参数(Z轴长度,Z速度,设定,采样率和持续时间)是可调的在不同的实验。


1米添加升AFM缓冲器Containing10毫米氯化钙2 -to腔室(图2) 。
 


D:\重新格式化\ 2020-2-7 \ 1902794--1332彭政802285 \图jpg \图2.jpg


图2.带有1 ml AFM缓冲溶液的AFM腔室的照片图像。黑圈显示了多蛋白的位置。


激光移动d提示的功能化镨OBE (图3) ,使用均分定理校准弹簧常数(卡帕)每个Cantilev中尔与溶液中的精确值在实验前。
 


D:\重新格式化\ 2020-2-7 \ 1902794--1332彭正802285 \图jpg \图3.jpg


图3. AFM实验中悬臂尖端的CCD图片。红色环位于D尖端的三角形。红色环指出了D尖端的激光辐射位置。


 


悬臂尖端连接到样品表面,并在此位置保持一会儿,以形成粘着蛋白/ 泊坞蛋白对,如图4,片段0和片段1所示。
以400 nm s -1 的恒定速度从表面收回悬臂,同时以4,096 Hz的采样率记录距离和悬臂挠度,如图4所示,第2段。
如图4的第3部分所示,将悬臂保持一会儿以放松粘着蛋白/ 泊坞蛋白对。
 


D:\重新格式化\ 2020-2-7 \ 1902794--1332彭正802285 \图jpg \图4.jpg


图4.关于AFM实验参数设置的对话框。一个AFM测量包含四个步骤。段0使悬臂向样品表面移动。段1使悬臂保持在段0达到的高度持续一段时间。段2撤回在此期间,表面上的悬臂和蛋白质展开。段3在持续时间内将悬臂保持在段2达到的高度。


 


 


资料分析


 


使用JPK数据处理选择力-延伸轨迹。
使用伊戈尔临6.12 (Wavemetrics )分析的数据痕迹。只有与数据路径包含所有特定的轮廓长度公差属于指纹蛋白结构域[ GB1(H18 nm)与CBM(H58牛米)] 和一种高断裂力从Cohesin-坞因子解离满足要求(超过200-300 PN )(图5 )。拟合曲线由蠕虫状链(WLC的)型号聚合物弹性与持效期长的〜0.4纳米。
使用高斯模型拟合展开力的直方图来确定最可能的展开力()和轮廓长度增量(< ∆ Lc >)的值。
 


D:\重新格式化\ 2020-2-7 \ 1902794--1332彭铮802285 \图jpg \图5.jpg


图5.序列控制的多蛋白Coh-Ub 4的典型力与距离的关系曲线,包含CBM展开,dockerin-cohesin 对破裂和Ub的四个拷贝的信号


 


菜谱


 


裂解缓冲液
50 mM Tris


150毫米氯化钠


pH值7.4


洗涤缓冲液
20 mM Tris


400毫米氯化钠


2 mM咪唑


pH值7.4


洗脱缓冲液
20 mM Tris


400毫米氯化钠


250 mM咪唑


pH值7.4


DTNB解决方案
0.5 mM 5,5'-二硫代双(2-硝基苯甲酸)


1毫米EDTA


720万尿素


TEV蛋白酶溶液
0.4  mg / ml的TEV蛋白酶


75毫米氯化钠


0.5毫米EDTA


25 mM Tris-HCl,pH 8.0


10%[v / v]甘油


AFM测量缓冲器
100 mM Tris


100毫米氯化钠


pH值7.4


铬酸
20克铬酸钾


M 40 大号超纯水


中号360 大号浓硫酸


M9 中号
0.4%葡萄糖


0.1 mM氯化钙2


2毫米MgSO 4


 


致谢


 


自然科学基金国民的中国(批准号:小号。21771103,21977047),自然科学基金江苏省(批准号:BK20160639)。该协议被修改从以前的工作“酶法生物合成和固定多蛋白验证在单分子水平(邓等人,2019)。


 


竞争利益


 


作者声明没有利益冲突。






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引用:Deng, Y., Shi, S., Zheng, B., Wu, T. and zheng, P. (2020). Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease. Bio-protocol 10(8): e3596. DOI: 10.21769/BioProtoc.3596.
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