Single vector system for efficient N-myristoylation of recombinant proteins in E. coli

Abstract

Background: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT). Prokaryotes are lacking endogenous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose.

Methodology/principal findings: Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium.

Conclusions/significance: The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.

Figure 1. SDS-PAGE of cells expressing Nef (A) or myristoylated Nef (myr.Nef) resulting from coexpression of Nef and hNMT (B).

Aliquots of the expression cultures were taken before as well as 1.5 and 5 hours after IPTG induction. The corresponding lanes are labeled with “B”, “1.5” and “5”, respectively. In addition, protein molecular weight marker (“M”) was applied.

Figure 2. Gel filtration chromatograms documenting the final purification step of Nef and myristoylated Nef (myr. Nef).

Protein samples were loaded onto a preparative HiLoad 16/60 Superdex 75 column, eluted with a flow rate of 1 ml/min and the absorbance at 280 nm was recorded. The elution profiles with maxima of the main peaks at around 61 ml indicate a monomeric state of Nef. Minor fractions eluting at around 44 and 51 ml reflect traces of aggregated protein and GST-PreScission protease, respectively.

Figure 3. SDS-PAGE of 2 ml fractions collected during the final gel filtration of myristoylated Nef.

Aliquots of the SEC fractions of myristoylated Nef (cf. Figure 2) were applied on a 15% SDS polyacrylamide gel followed by coomassie staining. SDS-PAGE revealed virtually pure Nef. Lanes are labeled with the elution volumes of the analyzed aliquots (57–71). Protein bands of the applied molecular weight marker (M) are labeled with the corresponding molecular weight in kDa.

Figure 4. Section of mass spectrum of myristoylated Nef.

Electrospray ionization mass spectrometry confirmed the successful myristoylation of Nef. Notably, no unmyristoylated Nef could be detected. The experimentally determined molecular weight of 24849 Da is in accordance with the theoretical value of 24851 Da.