Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded genetic code

Abstract

Tyrosine sulfation is an important post-translational modification that occurs in higher eukaryotes and is involved in cell-cell communication, viral entry and adhesion. We describe a protocol for the heterologous expression of selectively tyrosine-sulfated proteins in Escherichia coli through the use of an expanded genetic code that co-translationally inserts sulfotyrosine in response to the amber nonsense codon, TAG. The components required for this process, an orthogonal aminoacyl-tRNA synthetase specific for sulfotyrosine and its cognate orthogonal tRNA that recognizes the amber codon, are encoded on the plasmid pSUPAR6-L3-3SY, and their use, along with a simple chemical synthesis of sulfotyrosine, are outlined in this protocol. Specifically, the gene for a protein of interest is mutated such that the codon corresponding to the desired location of tyrosine sulfate is TAG. Co-transformation of an expression vector containing this gene and pSUPAR6-L3-3SY into an appropriate E. coli strain allows the overexpression of the site-specifically sulfated protein with high efficiency and fidelity. The resulting protein contains tyrosine sulfate at any location specified by a TAG codon, making this method significantly simpler and more versatile than competing methods such as in vitro enzymatic sulfation, chemical sulfation and peptide synthesis. Once the proper expression vectors are cloned, our protocol should allow the production of the desired sulfated proteins in <1 week.

Figure 1

(Schultz). (a) Map of the optimized pSUPAR6-L3-3SY plasmid expressing the components necessary for translational incorporation of sulfotyrosine in response to the TAG codon. (b) Synthesis of sulfotyrosine.

Figure 2

(Schultz) (a) FAS-TE protein (sTyr at position 2375) on a denaturing SDS-PAGE gel stained with Coomassie blue. (b) Sulfotyrosine-containing 412d and variants on a denaturing SDS-PAGE gel stained with Coomassie blue. Samples are: 412d with a modified sulfation pattern containing sTyr at V_H position 104 and Tyr at V_H position 107 (B1); 412d with the standard sulfation pattern corresponding to the human derived antibody containing sTyr at V_H positions 104 and 107 (B2); a variant of 412d from a selection for gp120 binding (unpublished data) with sTyr at V_H positions 104 and 107 (B3); a variant of 412d from a selection for gp120 binding (unpublished data) with sTyr at V_H positions 104 and 107 (B4).