Large scale modification of biomolecules using immobilized sortase A from Staphylococcus aureus

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• 作者：Max Steinhagen ^{msteinha@uni-leipzig.de} ; Katja Zunker ^{katja.zunker@uni-leipzig.de} ; Karoline Nordsieck ^{karoline.nordsieck@uni-leipzig.de} ; Annette G. Beck-Sickinger ; ^{beck-sickinger@uni-leipzig.de}
• 关键词：SrtA ; sortase A ; CDB ; chitin binding domain ; EPL ; expressed protein ligation ; CuAAC ; Cu(I) catalyzed azide/alkyne cycloaddition ; TAMRA ; 5(6)-carboxytetramethylrhodamine ; PEGA resin ; polyethyleneglycole based resin modified with a terminal amine group ; HPL
• 刊名：Bioorganic and Medicinal Chemistry
• 出版年：2013
• 出版时间：15 June, 2013
• 年：2013
• 卷：21
• 期：12
• 页码：3504-3510
• 全文大小：692 K

文摘

Recently, sortase A (SrtA) from Staphyloccus aureus moved into the focus of bioscience because of its ability to incorporate site specific modifications into proteins. The enzyme was mostly used to modify target proteins in an analytical scale, to study biomolecules in their cellular context. In this study, we show the applicability of SrtA mediated ligation for site specific modification of proteins in a large scale. Therefore, the reaction was first optimized using peptides and subsequently new reaction conditions were applied for the large scale biotinylation of interleukin-8. Furthermore, we established C-terminal immobilization of the SrtA on a PEG based resin and could demonstrate maintaining enzymatic activity. Immobilized SrtA significantly facilitates previous ligation protocols as the enzyme can be easily recycled. Also, the removal of excess reaction solution and the whole washing process is significantly accelerated, as centrifugation or filtration techniques can be applied instead of time-consuming chromatography steps.