Adenowo Abiola Fatimah, Masamba Priscilla, Qokoyi Ndibonani Kebonang, Oyinloye Babatunji Emmanuel, Kappo Abidemi Paul
Department of Medical Biochemistry, Faculty of Basic Medical Sciences, Lagos State University College of Medicine, PMB 21266, Ikeja, Lagos, Nigeria.
Molecular Biophysics and Structural Biology (MBSB) Group, Department of Biochemistry, University of Johannesburg, Kingsway Campus, Auckland Park 2006, South Africa.
Adv Pharm Bull. 2022 Mar;12(2):366-374. doi: 10.34172/apb.2022.035. Epub 2021 Jan 31.
Universal stress protein (USP) from , designated as G4LZI3, waspreviously hypothesised as a druggable target and vaccine candidate for human schistosomiasis.The purpose of this study is to characterize a purified recombinant G4LZI3 preliminarily forsubsequent structural characterization, which will provide baseline structural data for futurefunctional studies for the discovery, design and development of new schistosomal drugs for thetreatment, control and elimination of schistosomiasis. Restriction digest analysis of a GenScript-synthesised codon-optimised G4LZI3gene construct was carried out to ascertain its integrity and size. Thereafter, the pQE30-G4LZI3 construct was transformed into an M15 bacterial expression host. Transformed cellswere induced with isopropyl β-D-thiogalactoside for recombinant protein expression of anappreciable amount of pQE30-G4LZI3, which was subsequently purified with fast proteinliquid chromatography (FPLC) and a size exclusion chromatographic purification scheme.Preliminary biophysical characterization of the 6X His-tagged G4LZI3 was done to determineits secondary structure characteristics and protein stability. A molecular weight protein of 20.3 kDa was confirmed subsequent to restriction digestanalysis, while heterologous protein expression yielded a highly soluble and considerableamount of histidine-tagged G4LZI3 protein, which was successfully purified to homogeneity.Biophysical characterization indicated that the protein was well folded, heat-stable, had thefunctional groups and secondary structure composition required and was thus amenable tofurther structural characterization and determination. Biophysical characterization of purified G4LZI3 showed that further structuralstudies can be embarked upon on the use of G4LZI3 as a druggable target and possibly avaccine target against schistosomiasis via vaccinomics.
来自[具体来源未提及]的通用应激蛋白（USP），命名为G4LZI3，先前被假定为人类血吸虫病的可成药靶点和疫苗候选物。本研究的目的是对纯化的重组G4LZI3进行初步表征，以便后续进行结构表征，这将为未来关于发现、设计和开发用于治疗、控制和消除血吸虫病的新型血吸虫药物的功能研究提供基线结构数据。对金斯瑞合成的密码子优化的G4LZI3基因构建体进行限制性消化分析，以确定其完整性和大小。此后，将pQE30 - G4LZI3构建体转化到M15细菌表达宿主中。用异丙基β - D -硫代半乳糖苷诱导转化细胞，以实现可观量的pQE30 - G4LZI3的重组蛋白表达，随后用快速蛋白质液相色谱（FPLC）和尺寸排阻色谱纯化方案进行纯化。对6X组氨酸标签的G4LZI3进行初步生物物理表征，以确定其二级结构特征和蛋白质稳定性。限制性消化分析后确认分子量为20.3 kDa的蛋白质，而异源蛋白表达产生了高度可溶且大量的组氨酸标签的G4LZI3蛋白，并成功纯化至均一性。生物物理表征表明该蛋白质折叠良好、热稳定，具有所需的官能团和二级结构组成，因此适合进一步的结构表征和测定。纯化的G4LZI3的生物物理表征表明，可以通过疫苗组学将G4LZI3用作血吸虫病的可成药靶点以及可能的疫苗靶点进行进一步的结构研究。
