Probing HSP40 Proteins' C-Terminal Domain: Expression and Biophysical Insights

HSP40, also known as DNAJ, belongs to a family of co-chaperones responsible for delivering client proteins to HSP70 and stimulating HSP70 ATPase activity. The HSP70/DNAJ system plays a pivotal role in controlling proteostasis, which involves preventing protein aggregation and facilitating protein folding. DNAJ proteins are categorized into three main classes (A, B, and C) based on their structural domains. Class B DNAJs are homodimers consisting of a J-domain, a glycine-phenylalanine rich region, two β-barrel domains (CTDI and CTDII), and a dimerization domain. While these proteins are mainly involved in maintaining proteostasis for proteins linked to neurodegenerative diseases in humans, the specific role of each domain in this function remains unclear. The objective of this work was the expression and characterization of C-Terminal domain of DNAJB1 and DNABJ4 from human. The genes encoding DNAJB1 and DNAJB4 proteins were cloned into a pET151 D-TOPO vector and expressed in E. coli. After lysis, the proteins were recovered from the soluble fraction and purified using immobilized metal chelate affinity chromatography, followed by size exclusion chromatography. To assess their structural properties, circular dichroism (CD) spectropolarimetry and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) were employed for the characterization of DNAJB1 and DNAJB4 proteins. Our findings revealed successful expression of DNAJB1 and DNAJB4 within 4 hours at 37°C and 200 rpm agitation. Through the SEC-MALS technique, we confirmed that the expressed C-terminal domain of both DNAJB1 and DNAJB4 formed dimers. Additionally, circular dichroism analysis demonstrated that DNAJB1 and DNAJB4 were properly folded, as evidenced by their secondary structures. In conclusion, this study presents the successful expression, purification, and characterization of the C-terminal domain of DNAJB1 and DNAJB4 proteins. These findings contribute to our understanding of the roles played by these proteins in maintaining proteostasis, particularly concerning neurodegenerative diseases.