Serine/threonine ligation-assisted chemical synthesis of HMGA1a protein with site-specific post-translational modifications

Abstract

Summary Dissecting the function of proteins’ post-translational modifications (PTMs) is seriously hindered by the difficulty in obtaining the homogeneous protein with the PTMs of interest. Chemical protein synthesis offers a great potential to overcome this limitation. Here, a detailed protocol is introduced for chemical synthesis of HMGA1a protein with site-specific modifications via Ser/Thr ligation strategy, by which we can systematically study the function of the triple phosphorylation (3pSer) in the HMGA1a acidic tail. For complete details on the use and execution of this protocol, please refer to Wei et al. (2021) .

Protocol for semisynthesis of serotonylated histone H3 by rapid protein desulfurization in tandem with native chemical ligation

“… 15 Weigh 200 mg 2-Cl-(Trt)-Cl resin (0.6 mmol/ g ) into a peptide synthesis vessel similar to one from Wei et al. 16 Add 3 mL dimethylformamide (DMF) to the resin and drain it after 10 s. Repeat step ii for two more times. Add 3 mL dichloromethane (DCM) to the resin and drain it after 10 s. Repeat step iv for two more times.…”

“…Weigh 200 mg 2-Cl-(Trt)-Cl resin (0.6 mmol/ g ) into a peptide synthesis vessel similar to one from Wei et al. 16 …”

Oxidation and Phenolysis of Peptide/Protein C-Terminal Hydrazides Afford Salicylaldehyde Ester Surrogates for Chemical Protein Synthesis

“…The high abundance of N-terminal residues (Ser/Thr/Cys/Val) in native proteins and the good compatibility with the C-terminal residues in the SAL esters (with Lys, Glu, and Asp as exceptions) provide multiple choices for ligation sites. , In recent years, C-to-N − and N-to-C sequential STL strategies and the combined application of STL with NCL , have been developed through selective protection of N-terminal residues and the design of C-terminal SAL ester surrogates. With these developments, STL has been widely applied to obtain various protein and cyclic peptide products. − As a further advancement, protein semisynthesis is considered crucial for maximizing the synthetic efficiency. While expressed protein segments with N-terminal residues for STL/CPL (i.e., Ser/Thr) can be readily obtained, − the preparation of expressed proteins with C-terminal SAL esters, however, remains a significant challenge.…”