C-terminal modification and functionalization of proteins via a self-cleavage tag triggered by a small molecule

The precise modification or functionalization of the protein C-terminus is essential but full of challenges. Herein, a chemical approach to modify the C-terminus is developed by fusing a cysteine protease domain on the C-terminus of the protein of interest, which could achieve the non-enzymatic C-terminal functionalization by InsP6-triggered cysteine protease domain self-cleavage. This method demonstrates a highly efficient way to achieve protein C-terminal functionalization and is compatible with a wide range of amine-containing molecules and proteins. Additionally, a reversible C-terminal de-functionalization is found by incubating the C-terminal modified proteins with cysteine protease domain and InsP6, providing a tool for protein functionalization and de-functionalization. Last, various applications of protein C-terminal functionalization are provided in this work, as demonstrated by the site-specific assembly of nanobody drug conjugates, the construction of a bifunctional antibody, the C-terminal fluorescent labeling, and the C-terminal transpeptidation and glycosylation.


General Information and Procedures
High Performance Liquid Chromatography (HPLC).Method A: Analytical RP-HPLC was performed on a Thermo ultimate 3000 instrument with a C18 column (Agilent, 4 µm, 4.6 x 150 mm) at 40 o C. The column was eluted with a linear gradient of 2-90% acetonitrile containing 0.25% TFA in 30 min at a flow rate of 1 mL/min.Method B: Semi-preparative HPLC was performed on a Beijing ChuangXinTongHeng LC3000 (preparative) instrument with a preparative C-18 column (Waters, 5 µm, 19 x 250 mm).The column was eluted with a suitable gradient of aqueous acetonitrile containing 0.25% TFA at a flow rate of 8 mL/min.

Synthesis of peptide H-CDYKDDDK-OH
Peptide H-CDYKDDDK-OH was synthesized by standard solid phase peptide synthesis (SPPS) on 2-Chlorotrityl Chloride Resin (loading: 1.311 mmol/g).Amino acid couplings were done using 4 equivalents of amino acid with 4 equivalents of HATU and 8 equivalents of DIPEA in DMF.Fmoc removal was accomplished by incubating the resin with a 20% solution of piperidine in DMF for 15 min.
Synthesis of S3.To a stirred solution of CH3O-PEG24-COOH (58 mg, 0.05 mmol) in ACN was added N-hydroxysuccinimide (6.8 mg, 0.06 mmol) and EDC (7.9 µL, 0.06 mmol).The reaction mixture was stirred at room temperature for overnight.Then a solution of Fmoc-Lys(NH2)-OH (18.4 mg, 0.05 mmol) dissolved in ACN was added and the mixture was stirred at room temperature for 2 h.The mixture was purified by semi-preparative HPLC to get a white powder (78%).
Then the piperidine (100 µL) was added and the mixture was stirred at room temperature for 15 min.Then the solution was purified by semi-preparative HPLC to get S5 as a white powder (86%

Synthesis of cyclic peptide S7
To a stirred solution of peptide Ac-RGNCAYHRGKLVWCTYH-NH2 (5 mM) in DMSO was added H2O2 (10 mM) and NH3-H2O (100 mM) respectively, the mixture was stirred at room temperature for overnight.The mixture was purified by semi-preparative HPLC to get S7 as a white powder (90%

Synthesis of DBCO-tagged Trastuzumab
A solution of Trastuzumab (5 mg/mL), FcBP(Q10K)-TE-PEG4-DBCO (0.2 mM, 6 eq) and 3-maleimideopropionic acid (0.2 mM, 6 eq) in 50 mM PIPES buffer (pH 7.4) containing 20% DMF was incubated at 37 °C for 2 h.The progress of the reaction was monitored by ESI-TOF-MS.The reaction mixture was purified by affinity chromatography via protein A resin and concentrated by centrifugal filtration (30 kDa, Millipore).The buffer was exchanged for PBS buffer to give the DBCO-tagged antibody.

Synthesis of MTz-tagged Trastuzumab
A solution of Trastuzumab (5 mg/mL), FcBP(Q10K)-TE-PEG4-MTz (0.334 mM, 10 eq) and 3-maleimideopropionic acid (0.2 mM, 6 eq) in 50 mM PIPES buffer (pH 7.4) containing 20% DMF was incubated at 37 °C for 2 h.The progress of the reaction was monitored by ESI-TOF-MS.The reaction mixture was purified by affinity chromatography via protein A resin and concentrated by centrifugal filtration (30 kDa, Millipore).The buffer was exchanged for PBS buffer to give the MTz-tagged antibody.