期刊
NATURE PROTOCOLS
卷 8, 期 9, 页码 1808-1819出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2013.103
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资金
- Netherlands Organisation for Scientific Research
- US National Institutes of Health (NIH) [R01 AI087879]
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI087879] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [DP1GM106409] Funding Source: NIH RePORTER
Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-alpha, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3-4 d.
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