Cell-free synthesis of functionally active HSPB5.

ABSTRACT: Human αB-crystallin (HSPB5) is frequently modified post-translationally by UV radiation, oxidation, and age-associated processes, which complicates functional analyses of the protein using natural sources. Thus, determining the biological function of HSPB5 at the molecular structure level requires unmodified protein. Here, we employed an Escherichia coli cell-free protein synthesis system to prepare unmodified, functionally active human HSPB5. An S30 extract prepared from E. coli strain BL21 (DE3) was used for HSPB5 synthesis. The efficacy of protein synthesis was assessed by monitoring influencing factors, such as the concentrations of Mg²⁺ and other reaction mixture constituents, and by evaluating batch and/or dialysis synthesis systems. Chaperone-like activity of synthesized

SUBMITTER: Kojima R 

PROVIDER: S-EPMC7058722 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature