高效抗体标记由应变叠氮推广 - 炔环加成特定站点
Summary
Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).
Abstract
有可用的化学探针引入蛋白质以研究它们的结构和功能目前许多化学的工具。一种有用的方法是蛋白质缀合通过基因引入含有生物正交官能团的非天然氨基酸。这份报告介绍了位点特异性抗体偶联了详细的方案。该协议包括用于含叠氮氨基酸的遗传掺入，并通过应变促进的叠氮化物 – 炔环加成（SPAAC）的缀合反应的实验细节。该菌株促进的反应通过在生理pH和温度的反应的分子的简单混合进行，并且不需要另外的试剂如铜（I）离子和铜的螯合配体。因此，这种方法将是一般蛋白质偶联和抗体药物偶联物（ADC）的开发是有用的。
Introduction
由于在大肠杆菌 p -methoxyphenylalanine的遗传掺入报道，1 100多的非天然氨基酸（UAAs）已经被成功地结合到各种蛋白质。 1-3在这些UAAs，含生物正交功能基的氨基酸已被广泛研究，并代表的比例最大。在UAAs使用的生物正交功能团包括酮，4-叠氮基，5炔，6 cyclooctyne，7四嗪，8α，β不饱和酰胺，9降冰片烯，10 transcyclooctene，11和二环[6.1.0] -nonyne。 11虽然每个官能团都有其优点和缺点，在含叠氮基氨基酸被最广泛地用于蛋白质缀合。 p -Azidophenylalanine（AF），含叠氮基氨基酸之一，是容易得到的，它的掺入efficiency优异。含该氨基酸的突变蛋白可以通过铜催化环加成或与由SPAAC cyclooctynes炔进行反应。 12-20
近日，生物制药已在制药行业中引起极大关注。抗体-药物缀合物（ADC）是一类是有利的治疗性抗体的，由于其对人类癌症21和治疗靶向治疗能力 等疾病。超过50的ADC是目前在临床试验中，并且数量正在迅速增加。 ADC中的发展，需要考虑到最大限度的效力和减少副作用的许多因素。在这些因素中，一个高效的和位点特异性缀合反应以形成抗体之间形成共价键和一个药物是至关重要的。在缀合反应所需的效率和特异性可以通过缀合有生物正交功能组中的一个来实现是专门结合到抗体的非天然氨基酸。 22-26在这里，我们报告一个协议来位点特异性结合 AF成的抗体片段和缀合的突变体抗体片段与生物化学探针。
Protocol
1.质粒构建构建一个表达质粒（的pBAD-HerFab-L177TAG），将表达靶抗体基因（的pBAD-HerFab-WT）用他的6 -tag，并用琥珀密码子取代为亮氨酸177的密码子（TAG）27，用常规定点诱变技术。 见材料表 。 构建包含演进的tRNA 酪氨酸和氨酰tRNA合成酶（的aaRS）对所述基因另一个表达质粒（pEVOL-AFRS）。使用专门设计的质粒载体，pEVOL，为UAAs的有效结合。详细…
Representative Results
在这项研究中，抗体片段是位点特异性通过将一种含叠氮基氨基酸插入片段和突变体抗体片段与应变cyclooctyne反应用荧光团缀合（ 图1）。 HerFab被选为到其中的AF被合并为含叠氮基氨基酸的靶抗体片段。选择在HerFab残余物为带有AF更换，HerFab的X射线晶体结构进行了分析。对残余物30的重要要求包括来自抗体结合位点足够的距离，以尽量减少有效的缀…
Discussion
的非天然氨基酸遗传掺入蛋白质具有在用于蛋白质修饰的其它方法的几个优点。 1-3其中一个重要的优点是它的通用性的任何种类的蛋白质。原则上，存在选择目标蛋白质和该蛋白质的目标部位没有限制。然而，替换为结构上或功能上重要的残基的一个UAA可能导致改变靶蛋白的结构和功能。通常，暴露于溶剂，并且不与其他残基相互作用的残基被选择用于UAAs的掺入。因此，从一个高分辨率…
Divulgations
The authors have nothing to disclose.
Materials
| 1. plasmid Construction | |||
| plasmid pBAD_HerFab_L177TAG | optionally contain the amber stop codon(TAG) at a desired position. Ko, W. et al. Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. BKCS. 36 (9), 2352-2354, doi: 10.1002/bkcs.10423, (2015) | ||
| plasmid pEvol-AFRS | Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 (2), 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010) | ||
| DH10B | Invitrogen | C6400-03 | Expression Host |
| Plasmid Mini-prep kit | Nucleogen | 5112 | 200/pack |
| Agarose | Intron biotechnology | 32034 | 500g |
| Ethidium bromide | Alfa Aesar | L07482 | 1g |
| LB Broth | BD Difco | 244620 | 500g |
| 2. Culture preparation | |||
| 2.1) Electroporation | |||
| Micro pulser | BIO-RAD | 165-2100 | |
| Micro pulser cuvette | BIO-RAD | 165-2089 | 0.1cm electrode gap, pkg. of 50 |
| Ampicillin Sodium | Wako | 018-10372 | 25g |
| Chloramphenicol | Alfa Aesar | B20841 | 25g |
| Agar | SAMCHUN | 214230 | 500g |
| SOC medium | Sigma | S1797 | 100ML |
| 3. Expression and purification of HerFab-L177AF | |||
| 3.1 Expression of Herfab-L177AF | |||
| p-azido-L-phenylalanine (AF) | Bachem | F-3075.0001 | 1g |
| L(+)-Arabinose, 99% | Acros | 104981000 | 100g |
| Hydrochloric acid, 35~37% | SAMCHUN | H0256 | 500ml |
| 3.2 Cell lysis | |||
| Tris(hydroxymethyl)aminomethane, 99% | SAMCHUN | T1351 | 500g |
| EDTA disodium salt dihydrate, 99.5% | SAMCHUN | E0064 | 1kg |
| Sucrose | Sigma | S9378 | 500g |
| Lysozyme | Siyaku | 126-0671 | 1g |
| 3.3 Ni-NTA Affinity Chromatography | |||
| Ni-NTA resin | QIAGEN | 30210 | 25ml |
| Polypropylene column | QIAGEN | 34924 | 50/pack, 1ml capacity |
| Imidazole, 99% | SAMCHUN | I0578 | 1kg |
| Sodium phosphate monobasic, 98% | SAMCHUN | S0919 | 1kg |
| Sodium Chloride, 99% | SAMCHUN | S2907 | 1kg |
| 4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) | |||
| Cy5.5-ADIBO | FutureChem | FC-6119 | 1mg |
| 5. Purification of Labeled HerFab | |||
| Amicon Ultra 0.5 mL Centrifugal Filters | MILLIPORE | UFC500396 | 96/pack, 500ul capacity |
| 6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning | |||
| 1,4-Dithio-DL-threitol, DTT, 99.5 % | Sigma | 10708984001 | 10g |
| NuPAGE LDS Sample Buffer, 4X | Thermofisher | NP0007 | 10ml |
| MES running buffer | Thermofisher | NP0002 | 500ml |
| Nupage Novex 4-12% SDS PAGE gels | Thermofisher | NO0321 | 12well |
| Coomassie Brilliant Blue R-250 | Wako | 031-17922 | 25g |
| Typhoon 9210 variable mode imager | Amersham Biosciences |
References
- Wang, L., Schultz, P. G. Expanding the genetic code. Angew. Chem. Int. Ed. 44 (1), 34-66 (2004).
- Wu, X., Schultz, P. G. Synthesis at the interface of chemistry and biology. J. Am. Chem. Soc. 131 (35), 12497-12515 (2009).
- Liu, C. C., Schultz, P. G. Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79, 413-444 (2010).
- Wang, L., Zhang, Z., Brock, A., Schultz, P. G. Addition of the keto functional groupto the genetic code of Escherichia coli. proc. Natl. Acad. Sci. U.S.A. 100 (1), 56-61 (2003).
- Chin, J. W., et al. Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 124 (31), 9026-9027 (2002).
- Deiters, A., Schultz, P. G. In vivo incorporation of an alkyne into proteins in Esshcerichia coli. Bioorg. Med. Chem. Lett. 15 (5), 1521-1524 (2005).
- Plass, T., Milles, S., Koehler, C., Schultz, C., Lemke, E. A. Genetically encoded copper-free click chemistry. Angew. Chem. Int. Ed. 50 (17), 3878-3881 (2011).
- Seitchik, J. L., et al. Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with transcyclooctenes. J. Am. Chem. Soc. 134 (6), 2898-2901 (2014).
- Lee, Y. -. J., et al. A genetically encoded acrylamide fuctionality. ACS Chem. Biol. 8 (8), 1664-1670 (2013).
- Lang, K., et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal raction. Nat Chem. 4 (4), 298-304 (2012).
- Lang, K., et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels-Alder reactions. J. Am. Chem. Soc. 134 (25), 10317-10320 (2012).
- Jewett, J. C., Sletten, E. M., Bertozzi, C. R. Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones. J. Am. Chem. Soc. 132 (11), 3688-3690 (2010).
- Debets, M. F., et al. Azadibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloaddition. Chem. Commun. 46 (1), 97-99 (2010).
- Sletten, E. M., Bertozzi, C. R. From mechanism to mouse: a tale of two bioorthogonal reactions. Acc. Chem. Res. 44 (9), 666-676 (2011).
- Debets, M. F., et al. Bioconjugation with strained alkenes and alkynes. Acc. Chem. Res. 44 (9), 805-815 (2011).
- Mbua, N. E., Guo, J., Wolfert, M. A., Steet, R., Boons, G. -. J. Strain-promoted alkyne−azide cycloadditions (SPAAC) reveal new features of glycoconjugate biosynthesis. ChemBioChem. 12 (12), 1912-1921 (2011).
- Kuzmin, A., Poloukhtine, A., Wolfert, M. A., Popik, V. V. Surface functionalization using catalyst-free azide-alkyne cycloaddition. Bioconjug. Chem. 21 (11), 2076-2085 (2011).
- Yao, J. Z., et al. Fluorophore targeting to cellular proteins via enzymemediated azide ligation and strain-promoted cycloaddition. J. Am. Chem. Soc. 134 (8), 3720-3728 (2012).
- Hao, Z., Hong, S., Chen, X., Chen, P. R. Introducing bioorthogonal functionalities into proteins in living cells. Acc. Chem. Res. 44 (9), 742-751 (2011).
- Reddington, S. C., Tippmann, E. M., Jones, D. D. Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry. Chem. Commun. 48 (9), 8419-8421 (2012).
- Chari, R. V. J., Miller, M. L., Widdison, W. C. Antibody-drug conjugates: an emerging concept in cancer therapy. Angew. Chem. Int. Ed. 53 (15), 3751-4005 (2014).
- Tsao, M., Tian, F., Schultz, P. G. Selective staudinger modification of proteins containing p-Azidophenylalanine. ChemBioChem. 6 (12), 2147-2149 (2005).
- Brustad, E. M., Lemke, E. A., Schultz, P. G., Deniz, A. A. A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer. J. Am. Chem. Soc. 130 (52), 17664-17665 (2008).
- Milles, S., et al. Click strategies for single-molecule protein fluorescence. J. Am. Chem. Soc. 134 (11), 5187-5195 (2012).
- Jang, S., Sachin, K., Lee, h. J., Kim, D. W., Lee, h. S. Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis. Bioconjug. Chem. 23 (11), 2256-2261 (2012).
- Kazane, S. A., et al. Self-assembled antibody multimers through peptide nucleic acid conjugation. J. Am. Chem. Soc. 135 (1), 340-346 (2012).
- Ko, W., et al. Efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition. Bull. Korean Chem. Soc. 36 (9), 2352-2354 (2015).
- Young, T. S., Ahmad, I., Yin, J. A., Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 (2), 361-374 (2010).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976).
- Cho, H. S., et al. Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature. 421 (6924), 756-760 (2003).
- Kolb, H. C., Finn, M. G., Sharpless, K. Click chemistry : Diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40 (11), 2004-2021 (2001).
Citer Cet Article
Kim, S., Ko, W., Park, H., Lee, H. S. Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition. J. Vis. Exp. (118), e54922, doi:10.3791/54922 (2016).