Expression and purification of methionine aminopeptidases and N-terminal acetyltransferases

Methionine aminopeptidases (MetAPs) and N-terminal acetyltransferases (NATs) function co-translationally at the ribosome to enzymatically modify the emerging nascent chain. Eukaryotes express two types of MetAPs, namely MetAP1 and MetAP2, which can both carry out N-terminal methionine excision (NME) at the ribosome during translation. Following NME, the most abundant NAT, NatA, can acetylate the penultimate amino acid of the nascent chain, under regulation of the NatA inhibitor HypK. Alternatively, NatA can accommodate a second enzyme, called NAA50, to form the NatE complex capable of acetylating the initiator methionine. The abundant N-terminal modifications facilitated by MetAP1/2 and NatA/E-HypK impinge on protein function, interactions, lifetime and overall proteostasis. Robust and reliable methods for the expression and purification of MetAPs and NATs set the stage for targeted functional and structural studies of these enzymes. Established methods for the production of pure ribosome-associated enzymes and stochiometric complexes will be delineated in this chapter.