Multisite Clickable Modification of Proteins Using Lipoic Acid Ligase

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• 作者：Joseph G. Plaks ; Rebecca Falatach ; Mark Kastantin ; Jason A. Berberich ; Joel L. Kaar
• 刊名：Bioconjugate Chemistry
• 出版年：2015
• 出版时间：June 17, 2015
• 年：2015
• 卷：26
• 期：6
• 页码：1104-1112
• 全文大小：412K
• ISSN：1520-4812

文摘

Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), 伪-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein鈥損olymer, protein鈥搒ugar, and protein鈥揻atty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide鈥揳lkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.