Site-specific chemical modification of a glycoprotein fragment expressed in yeast

Abstract

Site-specific modification of glycoproteins has wide application in both biochemical and biophysical studies. This method describes the conjugation of synthetic molecules to the N-terminus of a glycoprotein fragment, viz., human immunoglobulin G subclass 1 fragment crystallizable (IgG1 Fc), by native chemical ligation. The glycosylated IgG1 Fc is expressed in a glycosylation-deficient yeast strain. The N-terminal cysteine is generated by the endogenous yeast protease Kex2 in the yeast secretory pathway. The N-terminal cysteine is then conjugated with a biotin thioester to produce a biotinylated, glycosylated IgG1 Fc using native chemical ligation.

Fig. 1

Schematic of synthesis of the biotin thioester 6. (i) Acetone; (ii) Ethanolamine; (iii) Biotin, DMF, DIC; (iv) 95% TFA.

Fig. 2

(a) Using PCR primer to encode a cysteine residue at the N-terminal of C-IgG1 Fc right next to the KR that is cleaved by Kex2 protease. (b) C-IgG1 Fc expressed in yeast is fused to a α-factor prepro leader sequence that directs the C-IgG1 Fc protein into the secretory pathway. The α-factor, which directs C-IgG1 Fc to be secreted, is cleaved from the fusion protein by the Kex2 protease in Golgi to generate the N-terminal cysteine.

Fig. 3

Purification gel of C-IgG1 Fc. Lane 1, molecular weight markers; lane 2, C-IgG1 Fc after Protein G column purification; lane 3, glycosylated C-IgG1 Fc after phenyl sepharose column purification.

Fig. 4

(a) ESI MS of heterogeneously glycosylated C-IgG1 Fc, calcd. masses for glycoforms containing 8-12 mannose residues: 26872, 27035, 27198, 27361, 27524 respectively, obs. 26873, 27034, 27198, 27360, 27523 respectively. (b) ESI MS of biotin modified heterogeneously glycosylated C-IgG1 Fc, calcd. masses for glycoforms containing 8-12 mannose residues: 27098, 27261, 27424, 27587, 27750 respectively, obs. 27098, 27259, 27423, 27585, 27748 respectively.

Fig. 5

(a) ESI MS of biotin modified deglycosylated C-IgG1 Fc, calcd. 25395, obs. 25392. (b) ESI MS of biotin modified Man5-C-IgG1 Fc, calcd. 26612, obs. 26609.