Cell-free synthesis of functionally active HSPB5

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 HSPB5 was assayed using alcohol dehydrogenase (ADH) under thermal stress. The amount of HSPB5 synthesized using the cell-free system depended significantly on the concentration of Mg²⁺ in the reaction mixture. Use of condensed S30 extract and increased levels of amino acids promoted HSPB5 production. Compared with the batch system, HSPB5 synthesis was markedly increased using the dialysis system. The construction vector played a critical role in regulating the efficacy of protein synthesis. HSPB5 synthesized using the cell-free system had a native molecular mass, as determined by mass spectrometry analysis. The co-presence of synthesized HSPB5 suppressed heat-associated denaturation of ADH. Human HSPB5 synthesized using the cell-free system thus retains functional activity as a molecular chaperone.