Long Recombinant Homogeneous Linkers for Dual Protein Conjugations, to Replace Heterogeneous PEG

3/11/2019 14:00 - 14:35

  • Dual protein conjugations
  • Orthogonally reactive linkers
  • Long homogenous linkers
  • Yeast expressed chemical linkers

Polyethyleneglycol (PEG) linkers are the classical tools for linking peptides and proteins, for example to other biomolecules or spectroscopic probes.

In some cases long linkers (> 5 kDa) are preferred for steric reasons or for pursuit of avidity effects, but available PEG linkers longer than 5 kDa are heterogeneous.

Long XTEN linkers (mixed sequences of GEDSTAP residues giving PEG-like properties) have been described with dual functionalities, thus enabling cross-linking of proteins etc.

However, XTEN sequences are rich in Ser and Glu, and we found that Ser/Glu have drawbacks for our use. Our main expression platform is based on yeast, and sequences with multiple Ser get partially O-mannosylated during yeast expression, thus leading to heterogeneous products. Also, we find that multiple Glu in linkers can increase the viscosity of solutions when high protein concentrations are needed, probably due to charge repulsion of multiple Glu's, and thus relatively stiff linkers. To solve these problems, we designed linkers based on repeats like (GQAP)n. Such sequences could be expressed as either long extensions from proteins (proteins with "recombinant PEG"), or (GQAP)n could be equipped with orthogonally reactive terminals to be used as linkers for dual conjugations.

We exemplify this with H-(GQAP)49-GQEP-Cys (MW 17848), which we expressed in high yield in yeast. The isolated product had mixed disulfides at Cys (paired with free Cys, glutathione or as homodimer), but we transformed the mixture to a material with homogeneous disulfide using excess 4-mercaptophenylacetic acid (64-69 % yield). The N-terminal amine was then transformed to azide (74-85 %), the disulfide was reduced to free Cys (60-86 %), and the dually reactive linker was conjugated to iodoacetyl-insulin in one end (Cys alkylation, 44-52 %), and conjugated as triazole in the azido end, to a trypsin inhibitor with alkyne functionality (47 % yield). Data for enzymatic stabilization of the insulin-trypsin inhibitor conjugate will be reported comparing to simple insulin/trypsin inhibitor mixtures. The described linkers could find broad use in dual peptide/protein conjugations.