Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids

Abstract

We have improved the incorporation of L-and D-forms of unnatural amino acid (UAA) N εthiaprolyl-L-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2-6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of N ε -Boc-L-Lysine (BocK) and N ε -propargyl-L-Lysine (PrK) by 2-5 folds compared to their free acid forms. Our work demonstrated that using methylester-capped UAAs for protein expression is a useful strategy to enhance the yields of UAA-incorporated proteins.

Linkage‐Specific Synthesis of Di‐ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK

“…We cultured the cells in media supplemented with 1 mM L-ThzK-OMe 1. As previously reported, [11] we could express the UB K48ThzK mutant with a yield of more than 20 mg per liter of cell culture, determined as the amount of protein eluted from the Ni-NTA column (Figure 1A). UB K48ThzK 2 was then treated with O-methylhydroxyamine to generate the UB K48CysK mutant 3, and the conversion of the thiazolidine ring to the 1,2-aminothiol functionality was confirmed by the decrease of the MW of UB as measured by MALDI-MS (Figure S1A-B).…”

“…We cultured the cells in media supplemented with 1 mM L‐ThzK‐OMe 1 . As previously reported, [11] we could express the UB K48ThzK mutant with a yield of more than 20 mg per liter of cell culture, determined as the amount of protein eluted from the Ni‐NTA column (Figure 1A).…”

“…Furthermore, Pyl‐RS has been engineered for incorporating ThzK (Scheme 1, 1 ), a thiazolidine conjugated Lys derivative, into proteins, and once incorporated, ThzK can be converted to CysK to provide the functionality for protein ligation [10] . We previously reported the use of methyl ester form of ThzK (L‐ThzK‐OMe) in E. coli cell culture for enhancing the yield of ThzK‐incorporated UB [11] . Here, we report that ThzK replacing individual Lys residues (K11, K48, and K63) in UB can be converted to CysK and used for the synthesis of diUB of defined linkages by protein ligation.…”

“…[10] We previously reported the use of methyl ester form of ThzK (L-ThzK-OMe) in E. coli cell culture for enhancing the yield of ThzK-incorporated UB. [11] Here, we report that ThzK replacing individual Lys residues (K11, K48, and K63) in UB can be converted to CysK and used for the synthesis of diUB of defined linkages by protein ligation. We further demonstrate that the Cys residue left by the ligation reaction at the diUB conjunction site can form disulfide conjugates with the catalytic Cys residues of E2 or HECT E3s or undergo dethiolation to generate a reactive dehydroalanine (Dha) functionality to trap DUBs.…”

Reprogramming natural proteins using unnatural amino acids

“…It has been demonstrated that methyl ester form of UAAs against free acid form showed much better yield; the improved yield is possibly due to the improved membrane permeability of the methyl ester forms of the UAAs to E. coli cells as compared to the free acid forms, even though the same tRNA synthetases and E. coli strains were used for incorporation. 129 A study in 2020 optimized the direct incorporation of a uorescent UAA (7-hydroxy-4-coumarin-yl)ethyl glycine, where MS methods conrm that the UAA is introduced at only one site and that there is limited precedent. 116 The biochemical implications of altering the cellular translation machinery, as employed in GCE-based tagging, have yet to be extensively explored.…”