Incorporation of Unnatural Amino Acids

Incorporation of Unnatural Amino Acids

The power of protein engineering has been largely expanded in recent years, creating new possibilities in the use of unnatural amino acids (UAA) to interrogate or construct reactivity, stability and spectral properties of engineered proteins.1 Experts at Profacgen provide the most up-to-date protein engineering technologies for incorporation of UAAs bearing bioorthogonal handles such as:

  • Azides for click reaction or Staudinger ligation;
  • Alkynes for click reaction;
  • Ketones for amine-based reactions;
  • Aryl halides for Palladium-catalyzed cross-coupling;
  • Alkenes for metathesis.

Profacgen offers two strategies for UAA incorporation: 1) Metabolic incorporation of azidohomoalanine or homopropargylglycine using native translational machinery; 2) Site-specific incorporation of UAAs by orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pairs.

Metabolic incorporation is a fast, sensitive, non-toxic and convenient way to introduce methionine analogs (azidohomoalanine and homopropargylglycine) that contain very small modifications during active protein synthesis. Azide- or alkyne-modified proteins are labeling by exogenous reagents using either click reaction or Staudinger ligation. Detection sensitivity in 1-D gels and Western blots is in the femtomole range and suitable for downstream spectroscopic analysis (e.g., LC/MS).2

Site-specific incorporation utilizes tRNA/aaRS pair to deliver UAAs in response to a nonsense or four-base (frameshift) codon. The engineered aaRS only recognize its cognate tRNA without interfering endogenous tRNAs. On the other hand, the orthogonal tRNA is not a substrate for native tRNA synthetases, but it can be recognized by the engineered synthetase to transfer the specific UAA. This approach has provided insights into questions that are otherwise impossible to solve.
Profacgen, relying on the advanced protein engineering platform and over a decade experience, offer a comprehensive set of UAA incorporation services to help our clients:

  • Design customized research plans;
  • Synthesize UAAs with proper bioorthogonal handles;
  • Perform metabolic or site-specific incorporation of UAAS;
  • Purify and characterize modified proteins.

For more information regarding Profacgen’s UAA incorporation service, please contact us or call 1-631-448-8149. Our customer service representatives are available 24 hours a day, Monday through Friday, to assist you.

References:

  1.  Lang, K.; et al. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chemical Reviews 2014, 114 (9): 4764-4806.
  2. Beatty K.E.; et al. Noncanonical amino acids in protein science and engineering. In: Köhrer C.; et al. (eds) Protein engineering. Nucleic acids and molecular biology 2009, (22), Springer, Berlin, Heidelberg

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