An engineered peptide tag-specific nanobody for immunoaffinity chromatography application enabling efficient product recovery at mild conditions

化学 洗脱 色谱法 亲和层析 配体(生物化学) 离解常数 吸附 选择性 重组DNA 生物化学 有机化学 催化作用 受体 基因
作者
Jun Ren,Hao Xiong,Chundong Huang,Fangling Ji,Lingyun Jia
出处
期刊:Journal of Chromatography A [Elsevier BV]
卷期号:1676: 463274-463274 被引量:4
标识
DOI:10.1016/j.chroma.2022.463274
摘要

Camelid-derived nanobody is emerging as a resourceful platform for developing immunoaffinity ligands for chromatography applications. Featured by high affinity and selectivity, BC2 nanobody (BC2-Nb), which can recognize a specific epitope tag (PDRKAAVSHWQQ, termed BC2T), is potential to be developed as a general tool for recombinant protein purification. However, excessively high affinity between binding partners makes the desorption of products less efficient and limits its application. Aiming to improve elution efficiency, structure-guided mutations of BC2-Nb were conducted to adjust the structural flexibility of its antigen-binding site. Six ligand variants were obtained with their binding affinity decreasing by about 100-fold. Among them, one mutated BC2-Nb named 44D was chosen to prepare immunoaffinity resin, and its adsorption and elution performance were well characterized. The site-directed mutation led to the equilibrium dissociation constant (KD) of BC2-Nb changing from 1.4 × 10-9 M to 1.4 × 10-7 M (44D). The resin using 44D as ligand retained a static binding capacity of 19.14 mg/mL toward BC2T-fused enhanced green fluorescent protein (eGFP-BC2T). Significantly improved elution efficiency was obtained with the mutated ligand. Protein recovery reached 94% at pH 3.5 for 44D-based resin, while the resin based on original BC2-Nb could only achieve its highest recovery of 84% at pH 2. In addition, a neutral elution condition (1 M arginine containing 50% propylene glycol, pH 7.4) was also found effective, which allowed a product recovery of 95%. The resin enabled direct capturing of eGFP-BC2T from bacterial lysates, and the one-step purification with the both elution conditions could achieve a product purity of more than 90%. This study provided a promising affinity ligand, and also proved the feasibility of controlling the elution process of nanobody-based affinity resin through the strategy of binding sites modification.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
PDF的下载单位、IP信息已删除 (2025-6-4)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
ZHAO完成签到,获得积分10
2秒前
星辰完成签到 ,获得积分10
3秒前
3秒前
ding应助科研通管家采纳,获得10
5秒前
Youdge应助科研通管家采纳,获得10
5秒前
上官若男应助科研通管家采纳,获得10
6秒前
6秒前
MchemG应助科研通管家采纳,获得10
6秒前
czh应助科研通管家采纳,获得10
6秒前
所所应助科研通管家采纳,获得10
6秒前
MchemG应助科研通管家采纳,获得10
6秒前
MchemG应助科研通管家采纳,获得10
6秒前
丘比特应助科研通管家采纳,获得10
6秒前
所所应助科研通管家采纳,获得10
6秒前
Lucas应助科研通管家采纳,获得10
6秒前
6秒前
6秒前
热心市民小红花应助哈哈采纳,获得50
7秒前
NexusExplorer应助Skuld采纳,获得10
7秒前
starry完成签到 ,获得积分10
8秒前
大个应助kaoyikaoli采纳,获得10
8秒前
BIT_lulu完成签到,获得积分10
9秒前
10秒前
Yueee发布了新的文献求助10
10秒前
Lucas应助张道恒采纳,获得10
11秒前
量子星尘发布了新的文献求助10
11秒前
WQ发布了新的文献求助10
12秒前
烟花应助momo采纳,获得10
12秒前
健康的青槐完成签到 ,获得积分10
12秒前
林白生完成签到,获得积分10
15秒前
Valiant发布了新的文献求助10
15秒前
16秒前
champion完成签到 ,获得积分10
17秒前
江川锦鲤完成签到,获得积分10
18秒前
aigj完成签到 ,获得积分10
19秒前
早起睡个回笼觉完成签到,获得积分10
20秒前
20秒前
22秒前
科研杰克完成签到,获得积分10
22秒前
秃顶双马尾完成签到,获得积分10
23秒前
高分求助中
A new approach to the extrapolation of accelerated life test data 1000
ACSM’s Guidelines for Exercise Testing and Prescription, 12th edition 500
‘Unruly’ Children: Historical Fieldnotes and Learning Morality in a Taiwan Village (New Departures in Anthropology) 400
Indomethacinのヒトにおける経皮吸収 400
Phylogenetic study of the order Polydesmida (Myriapoda: Diplopoda) 370
基于可调谐半导体激光吸收光谱技术泄漏气体检测系统的研究 350
Robot-supported joining of reinforcement textiles with one-sided sewing heads 320
热门求助领域 (近24小时)
化学 材料科学 医学 生物 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 遗传学 基因 物理化学 催化作用 冶金 细胞生物学 免疫学
热门帖子
关注 科研通微信公众号,转发送积分 3988920
求助须知:如何正确求助?哪些是违规求助? 3531290
关于积分的说明 11253247
捐赠科研通 3269903
什么是DOI,文献DOI怎么找? 1804830
邀请新用户注册赠送积分活动 882027
科研通“疑难数据库(出版商)”最低求助积分说明 809052