Engineering Protein Sequence Composition for Folding Robustness Renders Efficient Noncanonical Amino acid Incorporations

Abstract

A total synthesis of epothilone C (1) with concomitant formal synthesis of epothilone A is described, using immobilized reagents and scavengers to effect multistep synthetic transformations and purifications.

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…This approach was also successfully transferred and applied to analogs without fluorine atoms. 163 The successful incorporation of Hfl into leucine zipper protein A1 could be performed using a modified leucyl-tRNA synthetase of the host (E. coli). 164 The yield decreased to a fifth of the parent protein, but in the light of the related in vitro activation data this is reasonable.…”

Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in Escherichia coli

“…Prajna et al (2005) introduced fluorine atoms into EGFP through a residue-specific method. Although, F-Tyr was incorporated into EGFP (Thr65-Tyr66-Gly67), the chromophore and surrounding residues of our newly developed GFP variant (Gly65-Tyr66-Gly67) are entirely different from the reported EGFP variant (Nagasundarapandian et al 2010). Here, we prepared recombinant fluoro-protein through a residue-specific method.…”

“…Construction of plasmids and incorporation of F-Tyr into GFP DNA manipulations were performed as described earlier (Nagasundarapandian et al 2010). Tyrosine auxotrophic cells containing pQE80GFP construct were grown in M9 minimal medium supplemented with 0.4% (w/v) glucose, 0.1 mM CaCl 2 , 1 mM MgSO 4 , thiamine (35 lg/ml), 20 amino acids (40 mg/l), and ampicillin (10 lg/ml).…”

“…Recently, we developed an interesting GFP variant (Nagasundarapandian et al 2010) for the substitution of fluorine containing NCAA (fluorotyrosine, F-Tyr) in the place of tyrosine (L-Tyr) residues through a residue-specific method. Prajna et al (2005) introduced fluorine atoms into EGFP through a residue-specific method.…”

Manipulation of enzyme properties by noncanonical amino acid incorporation

“…Replacing proline residues of enhanced GFP (EGFP) with (4S)-fluoroproline resulted in enhanced solubility and higher refolding efficiency than that of the wild-type EGFP [43]. Very recently, Lee et al [48] discovered that GFP mutants with higher folding efficiency led to smaller reduction of cellular fluorescence upon the introduction of various methionine analogues: L-homopropargylglycine (12), L-azidohomoalanine (11), L-ethionine, L-norleucine (10), and L-methoxinine. These findings suggest that using primary protein sequences with high folding robustness enables us to reduce perturbation of protein structure upon residue-specific incorporation of NCAAs.…”