# Total Chemical Synthesis of RNF4 by Sequential Native Chemical Ligation: C‑To‑N Versus N‑To‑C Strategies

## TL;DR

Scientists chemically synthesized the RNF4 protein using two methods, finding that one approach simplified the process and could help study its role in cancer.

## Contribution

A reliable and simplified method for total chemical synthesis of RNF4 using N-to-C ligation is presented.

## Key findings

- RNF4 was successfully synthesized using sequential native chemical ligation with five peptide fragments.
- The N-to-C ligation strategy simplified the synthesis workflow and reduced purification steps.
- The synthesis method addresses challenges in creating large proteins for biological research.

## Abstract

RNF4, a RING-type
E3 ubiquitin ligase, targets polySUMOylated
proteins
for ubiquitination and subsequent proteasomal degradation. The ability
to chemically synthesize RNF4 will enable future studies of its structure
and biological function, particularly its role in degrading the oncoprotein
PML-RARα in acute promyelocytic leukemia. To achieve this, we
performed a total chemical synthesis of RNF4 using sequential native
chemical ligation. The presence of nine cysteine residues enables
stepwise ligation of five peptide fragments to assemble the full-length
protein. Two synthetic strategies were explored: the first employed
a convergent C-to-N ligation, while the second used an N-to-C ligation.
In the convergent C-to-N approach, cysteine residues were protected
with acetamidomethyl groups to prevent side reactions during ligation,
although this required multiple deprotection and purification steps.
Conversely, the N-to-C synthesis method proceeded efficiently without
cysteine protection, thereby simplifying the workflow and reducing
the number of purification steps. This research presents a reliable
and accessible method for the complete chemical synthesis of RNF4,
addressing significant challenges in synthesizing large proteins and
opening up new opportunities for future biological research.