Optimization of a recombinant BlaR-CTD protein formulation using the response surface methodology.

IF 5.7 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Journal of Biological Engineering Pub Date : 2024-01-11 DOI:10.1186/s13036-023-00399-9

Mohadeseh Haji Abdolvahab, Mojdeh Safari, Farkhonde Hasannejad, Nika Asefi, Alireza Salimi, Mahboobeh Nazari

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Abstract

The sequence of a carboxy-terminal of the β-lactam sensor-transducer protein (BlaR-CTD) from Bacillus licheniformis ATCC14580 was extracted from US7745193B2 patent and expressed in E. coli using pColdI vector as a soluble His-tag recombinant protein. In this study, several excipients were used to improve the stability of recombinant BlaR-CTD and obtain the optimal formulation for this protein using response surface methodology (RSM)/ Central Composite Design (CCD). Total protein concentration was measured by UV spectroscopy and the Bradford test. A total of 7 various factors were designed using four different excipients including Glycerol, Sucrose, Triton x-100, and Tween-20, and three different buffers like Tris, Borate, and PBS. By obtaining suitable excipients and buffer i.e. glycerol and sucrose, pH ranging from 7 to 9 were evaluated. The pH 7.62, glycerol 15.35%, and sucrose 152.52 mM were determined as the most suitable for improving the thermal stability of recombinant BlaR-CTD.

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利用响应面方法优化重组 BlaR-CTD 蛋白配方。

本研究从 US7745193B2 专利中提取了地衣芽孢杆菌 ATCC14580 的β-内酰胺传感-转导蛋白（BlaR-CTD）的羧基末端序列，并使用 pColdI 载体在大肠杆菌中表达为可溶性 His-tag 重组蛋白。本研究使用了几种辅料来提高重组 BlaR-CTD 的稳定性，并利用响应面方法学（RSM）/中央复合设计（CCD）获得了该蛋白的最佳配方。总蛋白浓度是通过紫外光谱和 Bradford 试验测定的。使用四种不同的辅料（包括甘油、蔗糖、Triton x-100 和 Tween-20）和三种不同的缓冲液（如 Tris、Borate 和 PBS），共设计了 7 个不同的因子。通过获得合适的辅料和缓冲液（即甘油和蔗糖），对 7 至 9 的 pH 值进行了评估。结果表明，pH 7.62、甘油 15.35% 和蔗糖 152.52 mM 最适于提高重组 BlaR-CTD 的热稳定性。

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来源期刊

Journal of Biological Engineering BIOCHEMICAL RESEARCH METHODS-BIOTECHNOLOGY & APPLIED MICROBIOLOGY

CiteScore

7.10

自引率

1.80%

发文量

审稿时长

17 weeks

期刊介绍： Biological engineering is an emerging discipline that encompasses engineering theory and practice connected to and derived from the science of biology, just as mechanical engineering and electrical engineering are rooted in physics and chemical engineering in chemistry. Topical areas include, but are not limited to: Synthetic biology and cellular design Biomolecular, cellular and tissue engineering Bioproduction and metabolic engineering Biosensors Ecological and environmental engineering Biological engineering education and the biodesign process As the official journal of the Institute of Biological Engineering, Journal of Biological Engineering provides a home for the continuum from biological information science, molecules and cells, product formation, wastes and remediation, and educational advances in curriculum content and pedagogy at the undergraduate and graduate-levels. Manuscripts should explore commonalities with other fields of application by providing some discussion of the broader context of the work and how it connects to other areas within the field.