Purification of Histidine Tagged High-Mobility Group Box 3 (HMGB3) Expressed in Escherichia Coli
High-mobility group box 3 (HMGB3) is a chromatin-associated architectural protein that plays a role in regulating and supporting cellular DNA-dependent processes, such as optimizing DNA replication, transcription, and repair (1). Previous research has shown HMGB3 is often overexpressed in cancerous cells, which makes it a potential, novel target for therapeutic interventions that can help increase the efficacy of existing cancer therapies (2). This study aimed to develop a protocol that can successfully express eukaryotic HMGB3 using the bacterial system Escherichia coli (E. coli) so that functional HMGB3 can be prepared for high-throughput screenings in order to determine potential inhibitors for future drug studies. Results revealed that the presence of rare codons within the DNA sequence of HMGB3 were the biggest obstacles to successful expression. After codon optimization, HMGB3 DNA was inserted into a pET-28b vector containing His-tag and expressed alongside GroEL/ES chaperone using the E. coli strain BL21 (DE3) competent cells. Purification was achieved utilizing Ni-NTA affinity chromatography and Mono-Q 10/10 anion-exchange chromatography in succession. The purity of HMGB3 protein was then assessed using SDS -PAGE gel.