Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Abstract

Staphylococcus aureus sortase A (SaSrtA) is widely used for site‐specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head‐to‐tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6‐fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6‐fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions.

Engineered Sortases in Peptide and Protein Chemistry

“…It was therefore attempted to improve the stability of the activity‐enhanced M4 mutant from the original yeast display screen(Table 1). [19,37] This mutant shows a more than 10 °C lower thermal melting temperature compared to wild‐type SaSrtA (48.6 vs 59.4 °C). Single site‐saturation mutagenesis of residues in the β6–β7 loop identified two mutations (R159N and K162P) that, when combined, led to a 6 °C higher stability of this new M6‐SrtA.…”

“…SrtA-M4/R159N/K162P LPxTG N-Gly-Gly + + yes increased thermostability of M4 [37] SrtA-P94H/A104T/E105D/G167E/Q172H LPxTG N-Gly-Gly + yes [21] SrtA-M3 LPxTG N-Gly-Gly + yes More DMSO resistent [24] Sortase mutants with altered specificity SrtA-F40 APxTG N-Gly-Gly À yes [25] SrtA-A1-22 APxTG N-Gly-Gly À yes [26] SrtA-F1-20 FPxTG N-Gly-Gly À yes [26] eSrtA(4S) LPxSG N-Gly-Gly + + yes engineered on SrtA-M5 template [27] eSrtA(2A) LAxTG N-Gly-Gly + + yes engineered on SrtA-M5 template [27] mSrt2A LAxTG N-Gly-Gly n.d. no engineered on eSrt(2A) template [28] Further sortase mutants Sa-SrtA-E105K/E108A LPxTG N-Gly-Gly (À ) no [29] Sa-SrtA-E105K/E108Q LPxTG N-Gly-Gly (À ) no [29] n.d. not determined in comparison to wild-type Sa-SrtA, + + strongly enhanced, + enhanced,À reduced, (À ) slightly reduced.…”

“…Further improvement of stability by 1.5°C was achieved by head-to-tail cyclization of the engineered variant. [37]…”

“…The relative activity of the M6 mutant was increased 58‐fold compared to wild‐type SaSrtA after pre‐incubation of the enzymes at 55 °C for 1 hour. Further improvement of stability by 1.5 °C was achieved by head‐to‐tail cyclization of the engineered variant [37] …”

Protein Macrocyclization for Tertiary Structure Stabilization

“…[ 46 , 47 , 48 ] More recently, the robustness of Sortase A has been improved, which can be expected to support the future application of Sortase A for head‐to‐tail cyclizations. [ 49 , 50 ] Butelase 1 from Clitoria ternetea recognizes a short peptide sequence (a3‐H−V; a3=asparagine (N) or aspartic acid (D)) and cleaves the amide bond between position a3 and histidine (H, 15 b , Figure 3 c). [51] To facilitate cyclizations, the first N‐terminal amino acid (a1) can be any amino acid, except for proline (P), while the second amino acid (a2) must be isoleucine (I), leucine (L), valine (V) or cysteine (C, 15 b , Figure 3 c).…”