Use of a Temporary “Solubilizing” Peptide Tag for the Fmoc Solid-Phase Synthesis of Human Insulin Glargine via Use of Regioselective Disulfide Bond Formation

Abstract

Solid-phase peptide synthesis has been refined to a stage where efficient preparation of long and complex peptides is now achievable. However, the postsynthesis handling of poorly soluble peptides often remains a significant hindrance to their purification and further use. Several synthetic schemes have been developed for the preparation of such peptides containing modifications to aid their solubility. However, these require the use of complex chemistry or yield non-native sequences. We describe a simple approach based on the use of penta-lysine "tags" that are linked to the C-terminus of the peptide of interest via a base-labile linker. After ready purification of the now freely solubilized peptide, the "tag" is removed by simple, brief base treatment giving the native sequence in much higher overall yield. The applicability of the method was demonstrated by the novel preparation of insulin glargine via solid-phase synthesis of each of the two chains--including the notoriously poorly soluble A-chain--followed by their combination in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. This approach should facilitate the development of new insulin analogues as well as be widely applicable to the improved purification and acquisition of otherwise poorly soluble synthetic peptides.

Polycationic amino acid tags enhance soluble expression of Candida antarctica lipase B in recombinant Escherichia coli

“…The 10-histidine tag at either the N-terminal or C-terminal could not increase the soluble expression of CalB, which was consistent with the previous report [10]. Polycationic peptides are known to enhance the solubility of proteins of interest [19] and favorable solubilizing propensity of polycationic peptides in solidphase peptide synthesis was demonstrated recently [20]. The same role of poly-arginine and poly-lysine tags in solubility improvement was reported in the overexpression of a bovine pancreatic trypsin inhibitor variant of BPTI-22 (a molecular weight of 5.9 kDa) [10].…”

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis

“…35 A less commonly used route for introducing semi-permanent C-terminal solubilizing tags entails C-terminal base-labile linkers—(e.g. esters) introduced either by Boc 36 (glycolic acid linker) or Fmoc-SPPS 37–39 (4-hydroxymethylbenzoic acid linker). An approach using the acid-labile phenylacetamido (PAM) linker has also been described 40 .…”

Preparation of canine relaxin by Fmoc-solid phase synthesis and regioselective disulfide bond formation within the A- and B-chains

“…Our approach of using efficient Fmoc-solid phase synthesis of suitably S-protected A-and B-chains followed by stepwise formation of the three disulfides has proven to be highly effective and robust with many assemblies of insulin-like peptides successfully accomplished [21,22,[29][30][31][32][33]. In order to acquire sufficient quantities of canine relaxin to undertake detailed structural and biological studies, we employed our approach (Figure 2A) and subsequently obtained highly purified peptide (Figures 2B and 2C) in overall yield of more than 6% relative to the starting crude B-chain.…”