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. 2010 Dec 23;5(12):e15801.
doi: 10.1371/journal.pone.0015801.

Targeted amino-terminal acetylation of recombinant proteins in E. coli

Matthew Johnson  1 , Arthur T Coulton, Michael A Geeves, Daniel P Mulvihill
Affiliations

Targeted amino-terminal acetylation of recombinant proteins in E. coli

Matthew Johnson et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of an E.coli NatB complex co-expression strain.
(A) Introns (black regions) were removed from genes encoding the NatB subunits Naa20 (white) and Naa25 (grey), and the subsequent cDNAs were each cloned into the same bacterial expression vector (pNatB). (B) Whole cell lysates from BL21-DE3 cells containing either pNatB alone (left), pTarget (encoding the target protein alone - middle), or both pNatB & pTarget (right) were separated by SDS-PAGE following IPTG induction and visualised using coomassie stain. These data confirmed the successful co-expression of the NatB complex and target proteins in E. coli.
Figure 2
Figure 2. Electron-spray mass-spectroscopy spectra for α-SkTm.
α-SkTm tropomyosin was purified from either E. coli BL21-pNatB cells (A) or E. coli BL21 cells (B). The undeconvolved mass charge envelope spectra show the purity of the proteins. Deconvolution of these data indicates that while ∼60% of the α-SkTm purified from BL21-pNatB cells is acetylated (additional 42 daltons mass), all of the Tm purified from standard BL21 cells remains unacetylated.
Figure 3
Figure 3. Electron-spray mass-spectroscopy spectra for S. pombe Cdc8.
Cdc8 was purified from E. coli BL21-pNatB cells. The un-deconvolved mass charge envelope spectra show the purity of the protein. Deconvolution of this data indicates that all of the Cdc8 purified from BL21-pNatB cells is acetylated.
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