Versatile protein biotinylation strategies for potential high-throughput proteomics.
We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo.
Lue
Rina Y P
RY
Department of Biological Sciences, National University of Singapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore.
Chen
Grace Y J
GY
Hu
Yi
Y
Zhu
Qing
Q
Yao
Shao Q
SQ
D019921:Biotinylation / Q000379:methods*; D002460:Cell Line; D002474:Cell-Free System; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040081:Protein Array Analysis; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D020349:Surface Plasmon Resonance
D005656:Fungal Proteins; D008164:Luminescent Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins