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Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems: toward functional protein arrays

Nature Protocols volume 1, pages 2386–2398 (2006)Cite this article

Abstract

This protocol details methodologies for the site-specific biotinylation of proteins using in vitro, in vivo and cell-free systems for the purpose of fabricating functional protein arrays. Biotinylation of recombinant proteins, in vitro as well as in vivo, relies on the chemoselective reaction between cysteine-biotin and a reactive thioester group at the C-terminus of a protein generated via intein-mediated cleavage. The cell-free system utilizes low concentrations of biotin-conjugated puromycin. Unlike other approaches that require tedious and costly downstream steps of protein purification, C-terminal biotinylated proteins can be captured directly onto avidin-functionalized slides from a mixture of other cellular proteins to generate the corresponding protein array. These methods were designed to maintain the integrity and activity of proteins in a microarray format, which potentially allows simultaneous functional assays of thousands of proteins. Assuming that the target proteins have been cloned into the expression vector, transformation of bacterial strain and growth of starter culture would take ∼2 days. Expression and in vitro protein purification and biotinylation will take ∼3 days whereas the in vivo method would take ∼2 days. The cell-free protein biotinylation strategy requires only 6–8 h.

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Figure 1
Figure 2
Figure 3: Schematic for cell-free biotinylation of proteins using 5′-Biotin-dc-Pmn.
Figure 4
Figure 5: Effect of C-terminal amino acid on on-column cleavage and biotinylation.
Figure 6: Analysis of samples collected at different steps during in vitro purification and on-column biotinylation of maltose binding protein–intein fusion using a chitin column.

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Authors and Affiliations

  1. Department of Biological Science, National University of Singapore, 3 Science Drive 3, Singapore, 117543, Republic of Singapore

    Souvik Chattopadhaya, Lay Pheng Tan & Shao Q Yao

  2. Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore, 117543, Republic of Singapore

    Shao Q Yao

  3. NUS MedChem Program of the Office of Life Sciences, National University of Singapore, 3 Science Drive 3, Singapore, 117543, Republic of Singapore

    Shao Q Yao

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  1. Souvik Chattopadhaya
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Correspondence to Shao Q Yao.

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Chattopadhaya, S., Tan, L. & Yao, S. Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems: toward functional protein arrays. Nat Protoc 1, 2386–2398 (2006). https://doi.org/10.1038/nprot.2006.338

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