Abstract：
Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with KD = 2.1 μM or KD = 1.6 μM toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and anti-hemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10-500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real-time and versatile monitoring of large-scale scFv production.
摘要：
单链片段变量(scFvs)，由通过肽接头连接在一起的可变重链和轻链组成，可以使用具有成本效益的细菌表达系统生产，使它们成为制药应用的有希望的候选人。然而,监测重组蛋白生产的通用方法尚未开发。在这里，我们报道了一种新型的基于抗scFv适体的生物传感系统，具有高特异性和多功能性。首先,通过指数富集，使用配体的竞争性系统进化筛选抗scFv适体，专注于独特的scFv特异性肽接头。我们选择了两个适体，P1-12和P2-63，对于抗人表皮生长因子受体（EGFR）scFv，KD=2.1μM或KD=1.6μM，分别。这两种适体可以选择性地结合scFv,但不结合抗EGFFv。此外，选择的适体识别具有不同CDR的各种scFvs，如抗4-1BB和抗血红蛋白scFv，表明他们识别独特的肽接头区域。使用基于方波伏安法的抗scFv适体开发了用于抗EGFRscFv的电化学传感器。因此，构建的传感器可以在稀释的培养基中监测10-500nM范围内的抗EGFRscFv浓度，用于细菌培养，涵盖了重组生产scFvs的预期浓度范围。这些成就有望实现药物scFv的连续监测传感器，这将使大规模scFv生产的实时和通用监测。
