Abstract
Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids (UNAAs), without the limitations of cell membrane permeability and the toxicity of unnatural components. Here, we describe a quick and simple method to synthesize unnatural proteins in CFPS system based on Escherichia coli crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii. The superfolder green fluorescent protein (sfGFP) and p-propargyloxyphenylalanine (pPaF) were used as the model protein and UNAA. The synthesis of unnatural sfGFPs was characterized by microplate spectrophotometer, affinity chromatography, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This protocol provides a detailed procedure guiding how to use the powerful CFPS system to synthesize unnatural proteins on demand.
Highlights
Protein is a vital class of biomolecules, and all living organisms employ it to fulfill essential structural, functional, and enzymatic roles to sustain life [1]
Many various unnatural amino acids (UNAAs) have been incorporated into proteins successfully by amber suppression with orthogonal translation systems (OTS) [1,8]
Unnatural proteins have been applied in protein modifications [9], biophysical probes [10], enzyMmeethoedsnPgroitnoce.e2r01in9,g2, 1[611], biomaterials [12] and biopharmaceutical protein production [21o3f ]1.2 The synthesis of unnatural proteins can be based on two systems
Summary
Protein is a vital class of biomolecules, and all living organisms employ it to fulfill essential structural, functional, and enzymatic roles to sustain life [1]. 20 natural amino acids can form proteins in a near-infinite number of combinations to make them have structural and functional diversity [1]. Expanding protein functions by incorporating unnatural amino acids (UNAAs) featuring novel functional groups is more important [4]. Unnatural orthogonal translation systems (OTS) are designed to incorporate UNAAs in vivo or in vitro [6,7]. Expanding the genetic code by incorporating UNAAs has become a significant opportunity in synthetic and chemical biology [3]. Incorporating UNAAs has become a significant opportunity in synthetic and chemical biology [3]. FiguFriegu1r.eE1x. pExepreimrimeennttaall ddeessigignn. (.a()aP)rePpraerpataiornatoifocnruodfecerxutrdacet;e(xb)treaxctrta;c(tbio)neoxfterxapcrteisosinonotfeemxpplartees;sion temp(cl)apteusri;fi(cca)tipoun roiffioc-taDtiNoAn;o(df)op‐utDrifiNcaAti;on(do)f paauRrSi;fi(cea) ctieolln-froeef paraoRteSin; (sey)nctheelsl‐isfrreeaecptiorno.tein synthesis 2re.1a.cMtioatner.ials
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
