Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins – A case study for Mefp-3 from Mytilus edulis

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2024-04-10 DOI:10.1016/j.pep.2024.106483

Constanze Zwies , Ángela María Vargas Rodríguez , Marcel Naumann , Franziska Seifert , Markus Pietzsch

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Abstract

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

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贻贝足蛋白质重组合成、DOPA修饰和分析的替代策略--贻贝Mefp-3的案例研究

由于含有 L-3,4-二羟基苯丙氨酸（DOPA），贻贝足蛋白（Mfps）具有与各种表面结合的独特特性。贻贝足蛋白-3（Mefp-3）是贻贝粘附斑中的几种蛋白之一。它在斑块-基质界面的定位表明，Mefp-3 在粘附过程中起着关键作用。因此，该蛋白适用于开发创新型生物粘合剂。然而，重组 Mfp-3 主要是在变性条件下从包涵体中纯化出来的。在此，我们介绍了一种利用 SUMO 融合技术获得可溶性无标记 Mefp-3 的稳健且可重复的方案。此外，我们还首次使用了一种来自Verrucomicrobium spinosum的微生物酪氨酸酶对Mefp-3中与肽结合的酪氨酸进行体外羟化。经 MALDI-TOF-MS 证实，高度羟基化的 Mefp-3 具有与商用胶水相当的优异粘合性能。这些结果证明了一种按需进行 DOPA 修饰的基于 Mfp3 的重组可溶性无标记蛋白质的协同和简化的高产率生产工艺。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.