Enhancing substrate specificity of microbial transglutaminase for precise nanobody labeling

IF 4.4 2区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Synthetic and Systems Biotechnology Pub Date : 2024-10-16 DOI:10.1016/j.synbio.2024.10.003
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Abstract

Streptomyces mobaraenesis transglutaminase (smTG) can be used for site-specific labeling of proteins with chemical groups. Here, we explored the use of modified smTG for the biosynthesis of nanobody-fluorophore conjugates (NFC). smTG catalyzes the conjugation of acyl donors containing glutamine with lysine-containing acceptors, which can lead to non-specific cross-linking. To achieve precise site-specific labeling, we employed molecular docking and virtual mutagenesis to redesign the enzyme's substrate specificity towards the peptide GGGGQR, a non-preferred acyl donor for smTG. Starting with a thermostable and highly active smTG variant (TGm2), we identified that single mutations G250H and Y278E significantly enhanced activity against GGGGQR, increasing it by 41 % and 1.13-fold, respectively. Notably, the Y278E mutation dramatically shifted the enzyme's substrate preference, with the activity ratio against GGGGQR versus the standard substrate CBZ-Gln-Gly rising from 0.05 to 0.93. In case studies, we used nanobodies 1C12 and 7D12 as labeling targets, catalyzing their conjugation with a synthetic fluorophore via smTG variants. Nanobodies fused with GGGGQR were successfully site-specifically labeled by TGm2-Y278E, in contrast to non-specific labeling observed with other variants. These results suggest that engineering smTG for site-specific labeling is a promising approach for the biosynthesis of antibody-drug conjugates.
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增强微生物转谷氨酰胺酶的底物特异性,实现精确的纳米抗体标记
链霉菌转谷氨酰胺酶(smTG)可用于用化学基团对蛋白质进行位点特异性标记。smTG 催化含谷氨酰胺的酰基供体与含赖氨酸的受体的连接,这可能导致非特异性交联。为了实现精确的位点特异性标记,我们采用了分子对接和虚拟诱变的方法,重新设计了酶对肽 GGGGQR(smTG 的非首选酰基供体)的底物特异性。从一个恒温高活性的 smTG 变体(TGm2)开始,我们发现单个突变 G250H 和 Y278E 显著增强了对 GGGQR 的活性,分别提高了 41% 和 1.13 倍。值得注意的是,Y278E 突变极大地改变了酶的底物偏好,对 GGGGQR 与标准底物 CBZ-Gln-Gly 的活性比从 0.05 上升到 0.93。在案例研究中,我们使用纳米抗体 1C12 和 7D12 作为标记目标,通过 smTG 变体催化它们与合成荧光团的连接。与其他变体的非特异性标记不同,与 GGGGQR 融合的纳米抗体成功地被 TGm2-Y278E 进行了位点特异性标记。这些结果表明,进行位点特异性标记的 smTG 工程是生物合成抗体-药物共轭物的一种可行方法。
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来源期刊
Synthetic and Systems Biotechnology
Synthetic and Systems Biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
6.90
自引率
12.50%
发文量
90
审稿时长
67 days
期刊介绍: Synthetic and Systems Biotechnology aims to promote the communication of original research in synthetic and systems biology, with strong emphasis on applications towards biotechnology. This journal is a quarterly peer-reviewed journal led by Editor-in-Chief Lixin Zhang. The journal publishes high-quality research; focusing on integrative approaches to enable the understanding and design of biological systems, and research to develop the application of systems and synthetic biology to natural systems. This journal will publish Articles, Short notes, Methods, Mini Reviews, Commentary and Conference reviews.
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