Earticle

초록

영어

Incorporating non canonical amino acids (NCAAs) into proteins is a powerful in vivo methodology that has been increasingly used. Currently, a number of NCAAs was incorporated into protein with the help the methodology such as reassignment of sense (residue specific incorporation) and non sense codon (site specific incorporation). Both methods balance one another in many ways. However, these two methods have a similar drawback in that they allow only incorporation of a single NCAA into the recombinant protein. Our group has developed an easy method to introduce two NCAAs containing different chemical moieties by coupling residue-specific and site-specific incorporation methods in a single protein. So far, all the techniques have demonstrated the possibility of introducing MNCAAs into a single protein; however, no studies have effectively utilized a multifunctional single protein in an effective way to show potential applications. Here, we used the MNCAA incorporation technique for self oriented immobilization (site- specific), to improve protein functionality, and for protein labeling (residue specific). For the purpose, we selected the surface exposed N-terminal residue (Lys15) of the green fluorescent protein (GFP) as a model protein. We introduced an amber codon (Lys15TAG) for the site- specific incorporation of L-DOPA using an evolved Methanococcus jannaschii tRNA/synthetase pairs and simultaneously (2S, 4S)-4-fluoroproline (4S-FPro) was selected for residue specific incorporation. In the next experiment, we prepared the MNCAAs protein with the methionine (Met) surrogate L-homopropargylglycine (L-HPG) (residue-specific incorporation) along with L -DOPA (site-specific incorporation) in GFP (GFPdphpg). The site-specific incorporation of L-DOPA into the protein will allow the protein to be immobilized in a controlled manner through Michael addition, the (2S, 4S)-4-fluoroproline (4S-F-Pro) was selected to improve protein stability. L-HPG contains an alkyne moiety and it will undergo a cycloaddition reaction with azide bearing molecules. We demonstrated that this method allows for site-specific attachment of the protein in a molecule for a subsequent labeling reaction. Incorporating MNCAA reduced the number of steps required for multiple labeling. We trust that the MNCAA protein engineering tool kit will occupy an important place in protein modification, as it offers numerous benefits and opportunities to develop synthetic biology research.

키워드

저자

• Kanagavel DEEPANKUMAR [ School of Biotechnology, Yeungnam University, Gyeongsan, South Korea. ]
• Hyungdon YUN [ School of Biotechnology, Yeungnam University, Gyeongsan, South Korea. ]

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발행기관

간행물

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• 간행물명

  한국생물공학회 학술대회

• 간기

  반년간

• 수록기간

  1985~2013

• 십진분류

  KDC 476 DDC 576