Sortase A-mediated chemoenzymatic synthesis of complex glycosylphosphatidylinositol-anchored protein

Abstract

Green fluorescent protein and a glycosylphosphatidylinositol (GPI) anchor containing the common core structure and a lipid chain were synthesized and then coupled together in the promotion of bacterial sortase A (SrtA), which was the first example for the synthesis of a full-size GPI-anchored protein by SrtA, demonstrating that this can be a generally useful method for GPI-anchored protein synthesis.

Figure 1

HPLC diagram of SrtA-medicated reaction of GPI anchor 1 and GFP 14. HPLC conditions: C-8 column (5 mm x 15 cm); flow rate: 1 mL/min; eluent: 10% to 60% aq. CH₃CN in 30 min; UV-Vis monitor at 220 nm.

Figure 2

SDS PAGE results of: molecular markers (Lane 1), the reaction mixture (Lane 2), isolated product 15 (Lane 3), GFP 14 (Lane 4), and SrtA (Lane 5).

Scheme 1

A general design for SrtA-catalyzed synthesis of GPI-anchored proteins

Scheme 2

Chemical synthesis of GPI anchor 1. Reaction conditions: (a) TMSOTf, toluene, 1,4-dioxane, −40 °C; (b) NaOMe, CH₂Cl₂, MeOH, 46% (2 steps); (c) TMSOTf, Et₂O, −40 °C; (d) NaOMe, CH₂Cl₂, MeOH, 76% (2 steps); (e) PivCl, pyridine, r.t.; then I₂, pyridine, H₂O, 74%; (f) 10% TFA in CH₂Cl₂, 69%; (g) (1) PivCl, pyridine, r.t.; (2) I₂, pyridine, H₂O, 72%; (h) H₂, 10% Pd(OH)₂/C, 88%.

Scheme 3

SrtA–mediated synthesis of GPI-GFP conjugate