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Abstract
One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co-expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.
Highlights
In eukaryotic cells up to 98% proteins are N-terminally acetylated [1]. In many of these cases the acetylation is required for normal function e.g. a-crystallin, S-adenosylmethionine decarboxylase, thymosin and components of the 26S proteasome regulatory particle [2,3,4,5]
Using a system based on the fission yeast NatB acetylation complex (Figure 1A), we have developed a method in which these are co-expressed together with target substrates in E. coli (Figure 1B)
Mammalian striated muscle a-Tropomyosin (Tm) is an evolutionarily conserved actin binding protein that is a substrate for the NatB complex [9,10]. This protein is readily expressed and purified from E. coli [11], and since the amino-terminal acetylation is essential for actin binding [11] it provides a simple test system for successful acetylation
Summary
In eukaryotic cells up to 98% proteins are N-terminally acetylated [1]. Amino terminal acetylation within eukaryotes is carried out by N-a-acetyltransferase (Nat) complexes and is thought to take place co-translationally at the ribosome [7]. 3 distinct classes of Nat complexes have been identified (NatA, B & C) [8], each composed of distinct catalytic and regulatory subunits. These complexes associate with specific and distinct target sequences at the amino terminus of elongating polypeptides
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