Purification of modified mammalian actin isoforms for in vitro reconstitution assays

Eur J Cell Biol. 2023 Sep 28;102(4):151363. doi: 10.1016/j.ejcb.2023.151363. Online ahead of print.ABSTRACTIn vitro reconstitution assays using purified actin have greatly improved our understanding of cytoskeletal dynamics and their regulation by actin-binding proteins. However, early purification methods consisted of harsh conditions to obtain pure actin and often did not include correct maturation and obligate modification of the isolated actin monomers. Novel insights into the folding requirements and N-terminal processing of actin as well as a better understanding of the interaction of actin with monomer sequestering proteins such as DNaseI, profilin and gelsolin, led to the development of more gentle approaches to obtain pure recombinant actin isoforms with known obligate modifications. This review summarizes the approaches that can be employed to isolate natively folded endogenous and recombinant actin from tissues and cells. We further emphasize the use and limitations of each method and describe how these methods can be implemented to study actin PTMs, disease-related actin mutations and novel actin-like proteins.PMID:37778219 | DOI:10.1016/j.ejcb.2023.151363

Source: European Journal of Cell Biology - October 1, 2023 Category: Cytology Authors: David J Kast Silvia Jansen Source Type: research