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Expression, purification, and phylogenetic analysis of MDIS1-INTERACTING RECEPTOR-LIKE KINASE1 (MIK1)
The Protein Journal ( IF 1.9 ) Pub Date : 2020-10-26 , DOI: 10.1007/s10930-020-09926-9
Krittin Trihemasava 1 , Sayan Chakraborty 1 , Kevin Blackburn 1, 2 , Guozhou Xu 1
Affiliation  

An abundance of protein structures has been solved in the last six decades that are paramount in defining the function of such proteins. For unsolved protein structures, however, predictions based on sequence and phylogenetic similarity can be useful for identifying key domains of interaction. Here, we describe expression and purification of a recombinant plant LRR-RLK ectodomain MIK1 using a modified baculovirus-mediated expression system with subsequent N-linked glycosylation analysis using LC–MS/MS and computational sequence-based analyses. Though highly ubiquitous, glycosylation site specificity and the degree of glycosylation influenced by genetic and exogenous factors are still largely unknown. Our experimental analysis of N-glycans on MIK1 identified clusters of glycosylation that may explicate the regions involved in MIK1 ectodomain binding. Whether these glycans are necessary for function is yet to be determined. Phylogenetic comparison using multiple sequence alignment between MIK1 and other LRR-RLKs, namely TDR in Arabidopsis thaliana, revealed conserved structural motifs that are known to play functional roles in ligand and receptor binding.



中文翻译:

MDIS1-相互作用受体样激酶1 (MIK1)的表达、纯化和系统发育分析

在过去的 60 年中,已经解决了大量蛋白质结构,这些结构对于定义此类蛋白质的功能至关重要。然而,对于未解决的蛋白质结构,基于序列和系统发育相似性的预测可用于识别相互作用的关键域。在这里,我们描述了重组植物 LRR-RLK 胞外域 MIK1 的表达和纯化,使用改良的杆状病毒介导的表达系统,随后使用 LC-MS/MS 和基于计算序列的分析进行 N 联糖基化分析。尽管高度普遍存在,糖基化位点特异性和受遗传和外源因素影响的糖基化程度仍然在很大程度上未知。我们对 MIK1 上 N-聚糖的实验分析确定了糖基化簇,这些簇可能解释了 MIK1 胞外域结合所涉及的区域。这些聚糖是否是功能所必需的还有待确定。MIK1 和其他 LRR-RLKs 之间使用多序列比对的系统发育比较,即 TDR拟南芥揭示了已知在配体和受体结合中发挥功能作用的保守结构基序。

更新日期:2020-10-30
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