Recombinant expression, purification, and structural analysis of two ectodomains of Syndecan-1.

外域 重组DNA 细胞生物学 劈理(地质) 融合蛋白 辛迪康1 蛋白多糖 化学 糖基化 细胞 生物 细胞外基质 生物化学 基因 古生物学 受体 断裂(地质)
作者
Austin R. Anderson,Gabriel A. Cook
出处
期刊:Protein Expression and Purification [Elsevier]
卷期号:201: 106170-106170 被引量:1
标识
DOI:10.1016/j.pep.2022.106170
摘要

Syndecan-1 (SDC-1) is an integral membrane heparin sulfate proteoglycan that is involved in inflammatory response, cell-signaling, cell proliferation, and numerous other cell-matrix interactions. Like the other members of the syndecan family, very little is known about structural conformations and dynamics of SDC-1. A majority of interactions occur through the extracellular ectodomain, therefore we have dedicated our research efforts to the study this specific portion of SDC-1. The ectodomain is often shed from the cell surface due to various stimuli. The released fragment has already been used as a useful biomarker for prognosis of some diseases and cancers. SDC-1 can be cleaved in different locations depending on the sheddase, generating soluble shed ectodomains that can be carried away in blood sera. In this study, we focus specifically on two main cleavage fragments that can be generated. We show the first successful expression and purification of recombinant SDC-1 ectodomains. Production of SDC-1 in E. coli allows the production of the core protein without risking heterogeneous post-translational modifications such as glycosylation, allowing a certain level of control over protein homogeneity that is not possible in mammalian expression. An expression vector was used to generate two different fusion proteins consisting of a His-tag and a TEV cleavage site for the removal of the fusion partner. SDS-PAGE was used to track the expression as well as the purification. Masses of the isolated proteins were determined using mass spectrometry and the purity and homogeneity were evaluated by solution NMR.
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