A cleavable self-aggregating tag scheme for the expression and purification of disulfide bonded proteins and peptides

Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists

“…The precipitated fusion protein was then resuspended in a solution at pH 6.2 (containing 0.7 M Na 2 SO 4 ) to induce C-terminal cleavage of Mtu ΔI-CM, releasing Di-GLP-1 or Tri-GLP-1 into the supernatant. We further purified Di-GLP-1 or Tri-GLP-1 using a two-step standard column purification, i.e ., IEC (ion exchange chromatogram, anion exchange resin) and SEC (size exclusion chromatogram, Superdex 75 resin). , …”

“…We observed that 67% of the icSAT-tagged GLP-1-SpyTag was prematurely cleaved, even when using the Mtu ΔI-CM variant (Figure C,D, line ES1). This premature cleavage was primarily induced by the N-terminal histidine of GLP-1, which is adjacent to the intein Mtu ΔI-CM and promotes cleavage at the C-terminus of Mtu ΔI-CM . Nevertheless, the Di- or Tri-SpyCatcher­(ΔN) scaffold could directly capture 49–61% of the prematurely cleaved GLP-1-SpyTag used for constructing Di-GLP-1 or Tri-GLP-1 (Figure C,D, line ES3).…”

“…We further purified Di-GLP-1 or Tri-GLP-1 using a two-step standard column purification, i.e., IEC (ion exchange chromatogram, anion exchange resin) and SEC (size exclusion chromatogram, Superdex 75 resin). 29,30 As shown in Figure 1C,D, the yields for icSAT-tagged GLP-1-SpyTag, Di-SpyCatcher(ΔN), and Tri-SpyCatcher(ΔN) were 203, 517, and 427 mg/L LB at the shake-flask scale, respectively. We observed that 67% of the icSAT-tagged GLP-1-SpyTag was prematurely cleaved, even when using the Mtu ΔI-CM variant (Figure 1C,D, line ES1).…”

“…This premature cleavage was primarily induced by the N-terminal histidine of GLP-1, which is adjacent to the intein Mtu ΔI-CM and promotes cleavage at the C-terminus of Mtu ΔI-CM. 30 Nevertheless, the Di-or Tri-SpyCatcher(ΔN) scaffold could directly capture 49−61% of the prematurely cleaved GLP-1-SpyTag used for constructing Di-GLP-1 or Tri-GLP-1 (Figure 1C,D, line ES3). Following an icSAT scheme (Table S2) and a two-step standard column purification (Figure S1), we Design and Preparation of GLP-1/GIP.…”

“…The protein expression method was modified from previous work. 30 Briefly, E. coli BL21(DE3) cells harboring the target plasmids were inoculated into lysogeny broth (LB) medium supplemented with 50 mg/L kanamycin and incubated at 37 °C with shaking (250 rpm). When the OD 600 reached 0.4−0.6, the incubation temperature was reduced to 18 °C, and after 15−30 min, isopropyl β-D-1thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM to initiate protein expression.…”