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Preparation of Recombinant Galectin-3 for Cancer Studies
用于癌症研究的重组半乳糖凝集素-3的制备   

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参见作者原研究论文

本实验方案简略版
Journal of Cellular Biochemistry
May 2006

Abstract

Galectin-3 is a member of a class of proteins termed Galectins, characterized by their ability to bind glycans containing β-galactose (Cummings and Liu, 2009). Galectin-3 binds preferentially to proteoglycans terminating with N-acetyllactosamine (LacNAc) chains (i.e., tandem repeats of galactose) (Newlaczyl and Yu, 2011). Galectin-3 is unique among the galectins in its chimeric structure. It shares a conserved carbohydrate recognition domain (CRD) with the other galectins, but has a long amino-terminal tail that is thought to be involved in protein aggregation. It can also form homodimers through its CRD (Cummings and Liu, 2009). Galectin-3 has been found to have diverse functions in tumorigenesis including: signaling, apoptosis inhibition, immune suppression, cell growth, and metastasis among others. Galectin-3 is frequently upregulated in cancers (Nangia-Makker et al., 2008). Its function largely depends on its expression and localization properties (Newlaczyl and Yu, 2011). Because of its many roles in cancer-associated processes, establishing a method for Galectin-3 production is valuable for further study of its functions in cancer. Here, we describe how Galectin-3 purification was achieved by cloning of the human Galectin-3 gene into pGEX-2T vector containing the gene for glutathione-S-transferase (GST) upstream of its cloning site. The Galectin-3 gene was cloned into this vector via restriction digests of both the plasmid and the Galectin-3 gene by restriction enzymes BamHI and EcoRI, followed by ligation of the two fragments. The resulting plasmid was then used to transform BL21, an Escherichia coli (E. coli) strain specialized for protein expression. Finally, we discuss how the GST fusion protein was isolated and the recombinant Galectin-3 protein was further purified from the GST.

Keywords: Cancer (癌症), Galectin (半乳糖凝集素), GST (消费税)

Part I. Protocol for GST-Gal3 purification (Harper and Speicher, 2011)

Materials and Reagents

  1. Microcentrifuge tubes (Denville Posi-Click Tubes, catalog number: C-2170 )
  2. 15 ml conical tubes (Thermo Fisher Scientific, Corning CentriStar, catalog number: 05-538-59A )
  3. 10 ml serological pipettes (Thermo Fisher Scientific, Corning Costar, catalog number: 4488 )
  4. One Shot® BL21 StarTM (DE3) Chemically Competent E. coli (Thermo Fisher Scientific, catalog number: C6010-03 )
  5. pGEX 2T-Gal3 vector (kindly provided by Dr. W. Stallcup, UCSD, CA)
  6. L-broth (Thermo Fisher Scientific, catalog number: BP1426-2 )
  7. Glutathione-Agarose Beads (Thermo Fisher Scientific, PierceTM, catalog number: 16100 )
  8. Protease Inhibitor Tablets, EDTA-free (Thermo Fisher Scientific, PierceTM, catalog number: 88665 )
  9. Ice
  10. Purified Water
  11. 10x PBS, diluted to 1x (Thermo Fisher Scientific, Hyclone, catalog number: BP3994 )
  12. Isopropyl-beta-D-thiogalactopyranoside (IPTG) (Gold Biotechnology, catalog number: 12481C25 )
  13. Tris-Cl (pH 7.5)
  14. 150 mM NaCl
  15. 0.75% CHAPS powder (AG. Scientific, catalog number: C-1019 )
  16. 0.75% CHAPS buffer (see Recipes)

Equipment

  1. Microfuge (Beckman Coulter, model: Microfuge 16 )
  2. 37 °C shaker (FormaOrbital Shaker)
  3. Shaker (GMI, Lab Companion, model: SK-300 Shaker )
  4. Rotating Platform (24 rpm) (Benchmark model: MiniMixer )
  5. Vortex (Thermo Fisher Scientific, catalog number: 12-812 )
    Note: Currently, it is “LabX, catalog number: 12-812”.
  6. Centrifuge (Beckman Coulter, model: Avanti J-20 XP )
  7. Centrifuge rotor (Beckman Coulter, model: JLA-16.250 )
  8. Centrifuge (Beckman Coulter, model: Allegra 25R )
  9. Centrifuge rotor (Beckman Coulter, model: TS-5.1-500 )
  10. 250 ml plastic centrifuge bottles (Sigma-Aldrich, Nalgene®, model: B1033 )
  11. 2 L flask (Corning, Pyrex®, model: No. 4980 )
  12. 10 ml serological pipettes (Corning, Costar®, catalog number: 4488 )

Procedure

  1. Transform BL21 bacteria with pGEX-2T-Gal3 expression vector, pick colonies and verify plasmid DNA with appropriate restriction digests (BamHI and EcoRI). The size of the plasmid is 5,697 bp; the size of the Galectin-3 insert is 753 bp (see map, Supplemental Figure 1).
  2. Use one colony to inoculate 3 ml clean, autoclaved Luria-broth and grow overnight at 37 °C at 250 rpm until OD600=1.0.
  3. In a 2 L flask, add 1,000 ml water. Measure 25 g Luria Broth and add to water in the flask. Autoclave before use (see step 2).
  4. Add 0.3 ml of the transformed bacteria to Luria Broth.
  5. Grow at 37 °C at 250 rpm for 4-5 h until OD600=0.6.
  6. Next, induce bacteria with 0.5 mM IPTG (119.2 mg) and allow growth on a shaker at room temperature at 200 rpm overnight for 16-18 h.
  7. Spin down bacteria into four, 250 ml plastic centrifuge bottles at 5,000 rpm for 15 min at 4 °C until pelleted. Discard supernatant. Place pellets on ice. (If you wish to stop here, the pellets may be stored at -80 °C until lysis. Prior to lysis, warm frozen pellets in 37 °C water bath for 10 min.)
  8. On ice, prepare CHAPS lysis buffer solution.
  9. Add two protease inhibitor tablets to 50 ml CHAPS lysis buffer solution and dissolve the tablets into the buffer by vortexing.
  10. On ice, add 12 ml CHAPS lysis buffer solution to each pellet.
  11. Break up the pellet by pipetting and vortexing until mixture is smooth. Vortex first to detach pellet from the wall of the centrifuge bottle, then use a 10 ml serological pipette to further disrupt the pellet by pipetting up and down to form a homogenous solution.
  12. Allow lysis to continue on ice for 30 min. Vortex every five minutes for the duration of the lysis (six times total).
  13. While bacterial lysis is taking place, add 2 ml glutathione-agarose bead slurry to 13 ml of purified water in 15 ml conical tube. Mix by inverting tube 2 to 3 times. Allow beads to swell for thirty minutes on ice.
  14. Distribute the bacterial lysate to microfuge tubes (about 1.5 ml lysate per tube, approximately 30 tubes). Alternatively, the lysate can be placed into a 50 ml centrifuge tube.
  15. Spin microfuge tubes at 14,000 rpm at 4 °C for 15 min. Supernatant should be clear. If using 50 ml centrifuge tube, spin at 3,500 rpm for 15 min at 4 °C. It is recommended, then, that the resulting supernatant be transferred to microfuge tubes and spun at 14,000 rpm for 10 additional minutes.
  16. Using a micropipette, separate supernatant from the pellets. Discard pellets.
  17. Pool supernatant from all microfuge tubes into two15 ml conical tubes.
  18. Gently aspirate excess water from swelled agarose-beads (beads should be settled at bottom of vial; if not centrifuge briefly for 10-20 sec without exceeding 1,000 rpm as high speeds can damage the beads).
  19. Add 10 ml CHAPS lysis buffer to beads to equilibrate. Mix by inverting the tube several times. Centrifuge briefly without exceeding 1,000 rpm.
  20. Gently aspirate excess buffer from the agarose beads.
  21. Add ~1 ml CHAPS lysis buffer to the agarose beads so that bead volume and buffer volume are equal (50% v/v). Mix beads and buffer by inverting the tube several times.
  22. Add 1 ml of the bead/buffer solution to each 15 ml conical tube containing bacterial lysis supernatant. Tip of pipette should be cut for beads to fit through.
  23. Mix beads and lysis supernatant by inverting the tube several times.
  24. Incubate overnight at 4 °C on slowly rotating platform (24 rpm).
  25. The next day, spin down supernatant and beads briefly without exceeding 1,000 rpm for ~10 sec at 4 °C to settle beads. Remove supernatant and save as it may still contain some of your protein and may be useful if further analysis is necessary.
  26. Wash GST-protein-bound beads by adding 14 ml 1x PBS to each tube. Mix by inverting. If beads are stuck at the bottom of the tube, gently flick the tube with your finger to mobilize the beads.
  27. Centrifuge briefly without exceeding 1,000 rpm to allow beads to settle. Remove 1x PBS, and wash again with another 14 ml 1x PBS.
  28. Remove 1x PBS. Beads should contain your GST-fusion protein.
  29. Assess the integrity and yield of your rGST-Gal3 protein with a Coomassie stained 12% polyacrylamide SDS-PAGE gel and/or Western blot (see Figure 2).
  30. Store beads in 50% v/v CHAPS buffer solution (~1 ml beads, ~1 ml CHAPS buffer) at 4 °C until further use or proceed with thrombin cleavage to separate Galectin-3.

Recipes

  1. 0.75% CHAPS buffer
    1.2 g (30 mM) Tris-Cl (pH 7.5)
    2.2 g (150 mM) NaCl
    1.9 g (0.75%) CHAPS powder
    Fill to 250 ml with water

Part II. Protocol for thrombin-mediated cleavage of GST from GST-Gal3

Materials and Reagents

  1. Microfuge tubes (DENVILLE SCIENTIFIC, catalog number: C-2170 )
  2. 375 U Thrombin (Merck Millipore Corporation, Calbiochem®, catalog number: 605195 )
  3. Glutathione-agarose beads bound with rGST-Gal3
  4. p-aminobenzamidine agarose beads [50% (V/V)] (Sigma-Aldrich, catalog number: A7155 )
  5. Tween-20 (Thermo Fisher Scientific, Fisher Bioreagent, catalog number: BP337-500 )
  6. Ice
  7. 10x PBS, diluted to 1x (Thermo Fisher Scientific, Hyclone, catalog number: BP3994 )
  8. Commercial Galectin-3 (Peprotech, catalog number: 450-38 , 50 ng)
  9. Anti-galectin-3 primary antibody (1:800 in PBST with 2.5% BSA, Santa Cruz, 20157)
  10. Chicken anti-rabbit secondary antibody (1:10,000 in PBST with 2.5% BSA, Santa Cruz, sc-2955)
  11. PBST (0.1% Tween 20) (see Recipes)

Equipment

  1. Rotating Platform (24 rpm) (Benchmark Scientific, model: MiniMixer )
  2. Microfuge (Beckman Coulter, model: Microfuge 16 )

Procedure

  1. On ice, add 500 μl PBST to 100 μl of the 50% v/v rGST-Gal3 bound agarose beads in CHAPS buffer in microfuge tubes (600 µl total volume/tube).
  2. On ice, prepare a thrombin solution by diluting 375 U of thrombin into 800 µl PBST.
  3. Add 400 µl of thrombin solution to each microfuge tube for a final volume of 1 ml per microfuge tube.
  4. Place tubes on rotating platform (24 rpm) and allow incubation overnight at room temperature.
  5. The next day, wash p-aminobenzamidine beads in 1x PBS by adding necessary amount of p-aminobenzamidine slurry (250 µl bead slurry per 100 µl rGST-Gal3 bound agarose beads) into a 15 ml centrifuge tube then filling the tube to 15 ml with 1x PBS. Mix by inverting then centrifuge briefly without exceeding 1,000 rpm.
  6. Remove 1x PBS then repeat wash with fresh 1x PBS.
  7. Remove PBS, then add enough fresh 1x PBS so that beads and 1x PBS form a 50% v/v solution.
  8. Centrifuge the bead/thrombin solution at 3,500 rpm for 1 min in tabletop microfuge. Remove supernatant and place into fresh 1.5 ml microfuge tubes.
  9. Add 250 µl p-aminobenzamidine agarose bead slurry to each supernatant-containing microfuge tube in order to remove thrombin from the solution.
  10. Incubate the p-aminobenzamidine beads with supernatant for 1.5 h at room temperature on rotating platform (24 rpm).
  11. Microfuge tubes at 3,500 rpm for ~30 sec.
  12. Collect supernatant. Proteins will be in supernatant while thrombin, attached to the beads, will be eliminated with the pellet.
  13. Assess supernatant protein composition with a Coomassie-stained SDS-PAGE and Western blot (Figures 1-2).


    Figure 1. Galectin-3 is successfully cleaved from GST through incubation with thrombin protease. Lanes 1-3 show increasing volumes of Galectin-3 (1 µl, 2 µl, 4 µl; approximately 150, 300, 600 ng) cleaved from GST through thrombin treatment. Lane 5 shows commercial Galectin-3. Samples were analyzed by SDS-PAGE on 12% polyacrylamide gel, followed by Western blot. Immunoblot was performed with Rabbit, anti-galectin-3 primary antibody (1:800 in PBST with 2.5% BSA), and chicken anti-rabbit secondary antibody (1:10,000 in PBST with 2.5% BSA).


    Figure 2. Analysis of thrombin cleavage of rGST-Gal3 by Coomassie-stained gel electrophoresis. Lane 1 shows positive control human Galectin-3 at ~25 kDa. Lane 2 shows GST-Gal3 without cleavage by thrombin. The size of the GST-Galectin3 fusion protein is approximately 50 kDa. Lanes 4-6 show increasing amounts of Galectin-3 cleaved from GST (0.94 µg, 1.88 µg, 3.75 µg as determined by Bradford Assay).

Recipes

  1. PBST (0.1% Tween 20)
    Add 100 µl of Tween-20 per 100 ml of 1x PBS

Acknowledgments

We thank Dr. William Stallcup (Sanford-Burnham Medical Research Institute; Cancer Center; La Jolla, CA USA) for providing the GST-Gal3 expression plasmids. This work was supported by R01 grant CA163722 from the NIH (to EGVM).

References

  1. Cummings, R. D., and Liu, F. T. (2009). Galectins. In: Varki, A., Cummings, R. D., Esko, J. D. , Freeze, H. H., Stanley, P., Bertozzi, C. R., Hart, G. W. and Etzler, M. E. (eds). Essentials of Glycobiology (2nd ed.). Cold Spring Harbor.
  2. Harper, S. and Speicher, D. W. (2011). Purification of proteins fused to glutathione S-transferase. Methods Mol Biol 681: 259-280.
  3. Nangia-Makker, P., Balan, V. and Raz, A. (2008). Regulation of tumor progression by extracellular galectin-3. Cancer Microenviron 1(1): 43-51.
  4. Newlaczyl, A. U. and Yu, L. G. (2011). Galectin-3--a jack-of-all-trades in cancer. Cancer Lett 313(2): 123-128.
  5. Wen, Y., Makagiansar, I. T., Fukushi, J., Liu, F. T., Fukuda, M. N. and Stallcup, W. B. (2006). Molecular basis of interaction between NG2 proteoglycan and galectin-3. J Cell Biochem 98(1): 115-127.

简介

Galectin-3是称为Galectins的一类蛋白质的成员,其特征在于它们结合含有β-半乳糖的聚糖的能力(Cummings和Liu,2009)。 Galectin-3优先结合以N-乙酰乳糖胺(LacNAc)链(即半乳糖的串联重复)终止的蛋白聚糖(Newlaczyl和Yu,2011)。 Galectin-3在其嵌合结构中的半乳凝素中是独一无二的。它与其他半乳凝素共享保守的碳水化合物识别结构域(CRD),但具有被认为参与蛋白质聚集的长氨基末端尾部。它也可以通过CRD形成同型二聚体(Cummings和Liu,2009)。已经发现Galectin-3在肿瘤发生中具有多种功能,包括:信号转导,凋亡抑制,免疫抑制,细胞生长和转移等。 Galectin-3通常在癌症中上调(Nangia-Makker等,2008)。其功能主要取决于其表达和定位性质(Newlaczyl和Yu,2011)。由于其在癌症相关过程中的许多作用,建立Galectin-3生产的方法对于进一步研究其在癌症中的功能是有价值的。在这里,我们描述如何通过将人Galectin-3基因克隆到其克隆位点上游的谷胱甘肽-S-转移酶(GST)基因的pGEX-2T载体中来实现Galectin-3纯化。通过限制酶BamHI和EcoRI,通过质粒和Galectin-3基因的限制性消化,将Galectin-3基因克隆到该载体中,然后连接两个片段。然后将所得质粒用于转化专门用于蛋白质表达的大肠杆菌(E.coli)菌株BL21。最后,我们讨论如何分离GST融合蛋白,并从GST进一步纯化重组Galectin-3蛋白。

关键字:癌症, 半乳糖凝集素, 消费税

第I部分。用于GST-Gal3纯化的方案(Harper和Speicher,2011)

材料和试剂

  1. 微量离心管(Denville Posi-Click Tubes,目录号:C-2170)
  2. 15ml锥形管(Thermo Fisher Scientific,Corning Centristar,目录号:05-538-59A)
  3. 10ml血清移液管(Thermo Fisher Scientific,Corning Costar,目录号:4488)
  4. One Shot ? BL21 Star TM (DE3)Chemically Competent 大肠杆菌(Thermo Fisher Scientific,目录号:C6010-03)
  5. pGEX 2T-Gal3载体(由Dr.W.Stallcup,UCSD,CA提供)
  6. L-broth(Thermo Fisher Scientific,目录号:BP1426-2)
  7. 谷胱甘肽 - 琼脂糖珠(Thermo Fisher Scientific,Pierce TM ,目录号:16100)
  8. 蛋白酶抑制剂片,无EDTA(Thermo Fisher Scientific,Pierce TM,目录号:88665)

  9. 纯化水
  10. 10x PBS,稀释至1x(Thermo Fisher Scientific,Hyclone,目录号:BP3994)
  11. 异丙基-β-D-硫代吡喃半乳糖苷(IPTG)(Gold Biotechnology,目录号:12481C25)
  12. Tris-Cl(pH7.5)
  13. 150mM NaCl
  14. 0.75%CHAPS粉末(AG.Science,目录号:C-1019)
  15. 0.75%CHAPS缓冲液(见配方)

设备

  1. Microfuge(Beckman Coulter,型号:Microfuge 16)
  2. 37℃振荡器(FormaOrbital Shaker)
  3. Shaker(GMI,Lab Companion,型号:SK-300振动器)
  4. 旋转平台(24 rpm)(基准型号:MiniMixer)
  5. Vortex(Thermo Fisher Scientific,目录号:12-812) 注意:目前,它是"LabX,目录号:12-812"。
  6. 离心机(Beckman Coulter,型号:Avanti J-20 XP)
  7. 离心转子(Beckman Coulter,型号:JLA-16.250)
  8. 离心机(Beckman Coulter,型号:Allegra 25R)
  9. 离心转子(Beckman Coulter,型号:TS-5.1-500)
  10. 250ml塑料离心瓶(Sigma-Aldrich,Nalgene ,型号:B1033)
  11. 2升烧瓶(Corning,Pyrex ,型号:No.4980)
  12. 10ml血清移液管(Corning,Costar ,目录号:4488)

程序

  1. 用pGEX-2T-Gal3表达载体转化BL21细菌,挑选菌落并用适当的限制性消化(BamHI和EcoRI)验证质粒DNA。质粒的大小为5,697bp;半乳凝素-3插入片段的大小为753bp(参见地图,补充图1 )。
  2. 使用一个菌落接种3ml清洁,高压灭菌的Luria-broth并在37℃下以250rpm生长过夜直到OD 600 = 1.0。
  3. 在2L烧瓶中,加入1,000ml水。测量25 g Luria Broth,加入烧瓶中的水中。在使用前进行高压灭菌(见第2步)。
  4. 将0.3ml转化的细菌加入Luria Broth中。
  5. 在37℃以250rpm生长4-5小时,直到OD 600 = 0.6
  6. 接下来,用0.5mM IPTG(119.2mg)诱导细菌,并允许在振荡器上在室温下以200rpm生长过夜,持续16-18小时。
  7. 将细菌在4℃下以5,000rpm旋转15分钟,直至沉淀,将细菌转入四个,250ml塑料离心瓶中。弃去上清液。将丸放在冰上。 (如果你想在这里停止,丸剂可以储存在-80℃直到裂解。在裂解前,将冷冻的丸粒在37℃水浴中温育10分钟)。
  8. 在冰上,制备CHAPS裂解缓冲液
  9. 将两种蛋白酶抑制剂片剂加入到50ml CHAPS裂解缓冲液溶液中,并通过涡旋将片剂溶解在缓冲液中
  10. 在冰上,向每个沉淀物中加入12ml CHAPS裂解缓冲液
  11. 通过吸移和涡旋破碎沉淀,直到混合物光滑。首先涡旋以从离心机瓶壁分离沉淀,然后使用10ml血清移液管通过上下吹打以进一步破坏沉淀,形成均匀的溶液。
  12. 允许裂解在冰上继续30分钟。裂解期间每五分钟涡旋一次(共六次)。
  13. 当细菌裂解发生时,在15ml锥形管中加入2ml谷胱甘肽 - 琼脂糖珠浆液到13ml纯水中。通过倒管混合2?3次。让珠子在冰上膨胀30分钟。
  14. 将细菌裂解物分配到微量离心管(每管约1.5ml裂解物,约30管)。或者,可将裂解物置于50ml离心管中。
  15. 旋转微量离心管在14,000 rpm在4℃15分钟。上清应清晰。如果使用50ml离心管,在4℃下以3500rpm旋转15分钟。然后建议将得到的上清液转移到微量离心管中并以14,000rpm离心10分钟。
  16. 使用微量移液管,从丸粒分离上清液。丢弃颗粒。
  17. 将来自所有微量离心管的上清液倒入两个15ml锥形管中
  18. 轻轻地从膨胀的琼脂糖珠上吸出过量的水(珠应该放置在瓶底部;如果不能快速离心10-20秒而不超过1000rpm,因为高速会损坏珠子)。
  19. 加入10ml CHAPS裂解缓冲液到珠子平衡。混合通过倒置管几次。简单离心,不超过1,000 rpm。
  20. 从琼脂糖珠中轻轻吸出过量的缓冲液
  21. 向琼脂糖珠中加入?1ml CHAPS裂解缓冲液,使得珠体积和缓冲液体积相等(50%v/v)。混合珠和缓冲液,通过倒置管几次
  22. 向每个含有细菌裂解上清液的15ml锥形管中加入1ml珠/缓冲溶液。移液器尖端应切割成珠子以适应。
  23. 通过倒置管数次混合珠和裂解上清液
  24. 在4℃下在缓慢旋转的平台(24rpm)上孵育过夜
  25. 第二天,将上清液和珠子短暂离心,不超过1,000rpm,在4℃下?10秒钟以沉淀珠子。除去上清液并保存,因为它可能仍含有一些蛋白质,如果需要进一步分析,可能有用
  26. 通过加入14毫升1×PBS到每个管洗涤GST蛋白结合珠。反转混合。如果珠子粘在试管底部,用手指轻轻摇动试管以移动珠子。
  27. 在不超过1,000rpm的条件下短暂离心以使珠沉降。取出1×PBS,并用另一只14 ml 1×PBS再次洗涤
  28. 删除1x PBS。珠子应该包含你的GST融合蛋白
  29. 使用考马斯染色的12%聚丙烯酰胺SDS-PAGE凝胶和/或蛋白质印迹(参见图2)评估rGST-Gal3蛋白的完整性和产量。
  30. 在4℃下将珠子储存在50%v/v CHAPS缓冲液(?1ml珠子,?1ml CHAPS缓冲液)中直至进一步使用或进行凝血酶切割以分离半乳凝素-3。

食谱

  1. 0.75%CHAPS缓冲液
    1.2g(30mM)Tris-Cl(pH7.5) 2.2克(150mM)NaCl 1.9g(0.75%)CHAPS粉末
    用水
    填充至250 ml

第二部分。凝血酶介导的GST从GST-Gal3切割的方案

材料和试剂

  1. Microfuge管(DENVILLE SCIENTIFIC,目录号:C-2170)
  2. 375 U凝血酶(Merck Millipore Corporation,Calbiochem ,目录号:605195)
  3. 与rGST-Gal3结合的谷胱甘肽 - 琼脂糖珠
  4. 对氨基苯甲脒琼脂糖珠[50%(V/V)](Sigma-Aldrich,目录号:A7155)
  5. Tween-20(Thermo Fisher Scientific,Fisher Bioreagent,目录号:BP337-500)

  6. 10x PBS,稀释至1x(Thermo Fisher Scientific,Hyclone,目录号:BP3994)
  7. 商业半乳凝素-3(Peprotech目录号:450-38,50ng)
  8. 抗半乳凝素-3第一抗体(在含2.5%BSA的PBST中1:800,Santa Cruz,20157)
  9. 鸡抗兔二抗(在含有2.5%BSA,Santa Cruz,sc-2955的PBST中为1:10,000)
  10. PBST(0.1%Tween 20)(参见配方)

设备

  1. 旋转平台(24rpm)(Benchmark Scientific,型号:MiniMixer)
  2. Microfuge(Beckman Coulter,型号:Microfuge 16)

程序

  1. 在冰上,在微量离心管中的CHAPS缓冲液(100μl总体积/管)中向100μl50%v/v rGST-Gal3结合的琼脂糖珠中加入500μlPBST。
  2. 在冰上,通过将375U凝血酶稀释到800μlPBST中来制备凝血酶溶液
  3. 向每个微量离心管中加入400μl的凝血酶溶液,每个微量离心管的最终体积为1 ml
  4. 将管放置在旋转平台(24 rpm),并允许在室温下孵育过夜
  5. 第二天,通过向15ml离心管中加入必需量的对氨基苯甲脒浆液(250μl珠浆液/100μlrGST-Gal3结合的琼脂糖珠),在1×PBS中洗涤对氨基苯甲脒珠,然后将管填充到15ml 1x PBS。通过倒置混合,然后短暂离心,不超过1000转/分
  6. 取出1×PBS,然后重复用新鲜的1×PBS清洗
  7. 取出PBS,然后加入足够的新鲜1×PBS,使珠和1×PBS形成50%v/v溶液。
  8. 在台式微量离心机中以3500rpm离心珠/凝血酶溶液1分钟。除去上清液,并放入新鲜的1.5毫升微量离心管。
  9. 向每个含有上清液的微量离心管中加入250μl对氨基苯甲脒琼脂糖珠浆液,以从溶液中除去凝血酶。
  10. 在室温下在旋转平台(24rpm)上将对氨基苯甲脒珠与上清液孵育1.5小时。
  11. Microfuge管在3500rpm,?30秒。
  12. 收集上清液。蛋白质将在上清液中,而附着在珠粒上的凝血酶将被沉淀物消除
  13. 用考马斯染色的SDS-PAGE和Western印迹评估上清液蛋白质组成(图1-2)

    图1.通过与凝血酶蛋白酶温育,半乳凝素-3从GST成功裂解。 泳道1-3显示通过凝血酶处理从GST切割的Galectin-3(1μl,2μl,4μl;约150,300,600ng)的体积增加。泳道5显示商业半乳凝素-3。通过在12%聚丙烯酰胺凝胶上的SDS-PAGE,随后通过蛋白质印迹分析样品。用兔,抗半乳凝素-3一抗(在含2.5%BSA的PBST中1:800)和鸡抗兔二抗(在含2.5%BSA的PBST中1:10000)进行免疫印迹。


    图2.通过考马斯染色的凝胶电泳分析rGST-Gal3的凝血酶切割。泳道1显示?25kDa的阳性对照人半乳凝素-3。泳道2显示没有被凝血酶切割的GST-Gal3。 GST-Galectin3融合蛋白的大小约为50kDa。泳道4-6显示从GST切割的半乳凝素-3的增加量(0.94μg,1.88μg,3.75μg,通过Bradford Assay测定)。

食谱

  1. PBST(0.1%Tween 20)
    每100 ml 1x PBS加入100μlTween-20

致谢

我们感谢William Stallcup博士(Sanford-Burnham医学研究所;癌症中心; La Jolla,CA USA)提供GST-Gal3表达质粒。这项工作是由R01授予CA163722从NIH(到EGVM)支持。

参考文献

  1. Cummings,R.D。,和Liu,F.T。(2009)。半乳凝素。 In:Varki,A.,Cummings,R.D.,Esko,J.D.,Freeze,H.H.,Stanley,P.,Bertozzi,C.R.,Hart,G.W.and Etzler,M.E。 Essentials of Glycobiology(第二版)。冷泉港。
  2. Harper,S.and Speicher,D.W。(2011)。 纯化与谷胱甘肽S-转移酶融合的蛋白质 /em> 681:259-280。
  3. Nangia-Makker,P.,Balan,V。和Raz,A。(2008)。 通过细胞外半乳凝素-3调节肿瘤进展。癌症微环境em> 1(1):43-51。
  4. Newlaczyl,A.U。和Yu,L.G。(2011)。 Galectin-3 - 一种癌症中的全能性。 < em> Cancer Lett 313(2):123-128。
  5. Wen,Y.,Makagiansar,I.T.,Fukushi,J.,Liu,F.T.,Fukuda,M.N.and Stallcup,W.B。(2006)。 NG2蛋白多糖和半乳凝素-3之间的相互作用的分子基础 J Cell Biochem。98(1):115-127。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tyler, K., Lee, S. and Van Meir, E. G. (2016). Preparation of Recombinant Galectin-3 for Cancer Studies. Bio-protocol 6(1): e1696. DOI: 10.21769/BioProtoc.1696.
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