应用RAD多肽展示体系制备抗hCG类抗体分子
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摘要
应用基于激烈火球菌Pyrococcus furiosus重组酶RadA的ATP酶结构域(RAD骨架)的多肽展示体系,通过嫁接人绒毛膜促性腺激素(hCG)结合多肽,制备抗hCG类抗体分子。通过合成hCG结合多肽插入RAD多肽展示位点的类抗体基因,成功构建了pET30a-RAD/hCGBP-sfGFP原核表达载体,在大肠杆菌中诱导蛋白表达,分离、纯化获得类抗体蛋白,通过亲和吸附-GFP荧光检测方法测定类抗体对hCG的结合活性,并与应用单域抗体通用骨架制备的嫁接抗体比较活性差异。结果显示,RAD类抗体分子对hCG分子具有较高的亲和性和特异性,显著优于单域嫁接抗体,并与商业单克隆抗体的活性相当;同时,利用RAD多肽展示骨架制备的抗hCG类抗体,具有较高的生化稳定性,是一种具有应用潜力的抗体替代分子。
By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector(pET30 a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector(pET30 a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
引文
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[13]Tiede C,Tang AAS,Deacon SE,et al.Adhiron:a stable and versatile peptide display scaffold for molecular recognition applications.Protein Eng Des Sel,2014,27(5):145-155.
[14]Zhang HX,Jiang SS,Zhang XF,et al.Protein kinase CK2αcatalytic subunit is overexpressed and serves as an unfavorable prognostic marker in primary hepatocellular carcinoma.Oncotarget,2015,6(33):34800-34817.
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[19]?krlec K,?trukelj B,Berlec A.Non-immunoglobulin scaffolds:a focus on their targets.Trends Biotechnol,2015,33(7):408-418.
[20]Nand KN,Gupta JC,Panda AK,et al.Development of a recombinant hCG-specific single chain immunotoxin cytotoxic to hCG expressing cancer cells.Protein Expr Purif,2015,106:10-17.
[21]Pellegrini L,Yu DS,Lo T,et al.Insights into DNArecombination from the structure of a RAD51-BRCA2complex.Nature,2002,420(6913):287-6993.
[2]Elliott MM,Kardana A,Lustbader JW,et al.Carbohydrate and peptide structure of theε-andβ-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma.Endocrine,1997,7(1):15-32.
[3]Cole LA,Shahabi S,Butler SA,et al.Utility of commonly used commercial human chorionic gonadotropin immunoassays in the diagnosis and management of trophoblastic diseases.Clin Chem,2001,47(2):308-315.
[4]Jewell EL,Aghajanian C,Montovano M,et al.Association of?-hCG surveillance with emotional,reproductive,and sexual health in women treated for gestational trophoblastic neoplasia.J Womens Health(Larchmt),2018,27(3):387-393.
[5]Peng J,Wang Q,Cheng XL,et al.Preparation of anti-hCG single domain antibody by antibody grafting technique using an antigen-binding peptide.Chin J Biotech,2018,34(4):569-577(in Chinese).彭静,王琼,程小玲,等.利用抗原结合多肽嫁接抗体技术制备抗hCG单域抗体.生物工程学报,2018,34(4):569-577.
[6]Liang AH,Li CN,Li D,et al.A facile and sensitive peptide-modulating graphene oxide nanoribbon catalytic nanoplasmon analytical platform for human chorionic gonadotropin.Int J Nanomedicine,2017,12:8725-8734.
[7]Rossmann M,Greive SJ,Moschetti T,et al.Development of a multipurpose scaffold for the display of peptide loops.Protein Eng Des Sel,2017,30(6):419-430.
[8]Lolli G,Pinna LA,Battistutta R.Structural determinants of protein kinase CK2 regulation by autoinhibitory polymerization.ACS Chem Biol,2012,7(7):1158-1163.
[9]Scott DE,Ehebauer MT,Pukala T,et al.Using a fragment-based approach to target protein-protein interactions.Chembiochem,2013,14(3):332-342.
[10]Shin DS,Pellegrini L,Daniels DS,et al.Full-length archaeal Rad51 structure and mutants:mechanisms for RAD51 assembly and control by BRCA2.EMBOJ,2003,22(17):4566-4576.
[11]Wu Y,Qian XG,He YJ,et al.Crystal structure of an ATPase-active form of Rad51 homolog from Methanococcus voltae:Insights into potassium dependence.J Biol Chem,2005,280(1):722-728.
[12]Moody IS,Verde SC,Overstreet CM,et al.In vitro evolution of an HIV integrase binding protein from a library of C-terminal domainγS-crystallin variants.Bioorg Med Chem Lett,2012,22(17):5584-5589.
[13]Tiede C,Tang AAS,Deacon SE,et al.Adhiron:a stable and versatile peptide display scaffold for molecular recognition applications.Protein Eng Des Sel,2014,27(5):145-155.
[14]Zhang HX,Jiang SS,Zhang XF,et al.Protein kinase CK2αcatalytic subunit is overexpressed and serves as an unfavorable prognostic marker in primary hepatocellular carcinoma.Oncotarget,2015,6(33):34800-34817.
[15]Benyamini H,Friedler A.Using peptides to study protein-protein interactions.Future Med Chem,2010,2(6):989-1003.
[16]Katz C,Levy-Beladev L,Rotem-Bamberger S,et al.Studying protein-protein interactions using peptide arrays.Chem Soc Rev,2011,40(5):2131-2145.
[17]Yan JR,Li GH,Hu YH,et al.Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications.J Transl Med,2014,12:343.
[18]Reverdatto S,Burz DS,Shekhtman A.Peptide aptamers:development and applications.Curr Top Med Chem,2015,15(12):1082-1101.
[19]?krlec K,?trukelj B,Berlec A.Non-immunoglobulin scaffolds:a focus on their targets.Trends Biotechnol,2015,33(7):408-418.
[20]Nand KN,Gupta JC,Panda AK,et al.Development of a recombinant hCG-specific single chain immunotoxin cytotoxic to hCG expressing cancer cells.Protein Expr Purif,2015,106:10-17.
[21]Pellegrini L,Yu DS,Lo T,et al.Insights into DNArecombination from the structure of a RAD51-BRCA2complex.Nature,2002,420(6913):287-6993.