Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements

Abstract

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.

Figure 1

Schematic representation of vectors for expression of MAPPHis and NMT. (A) the plasmid based on the pET22b vector coding gene for MAPPHis used in the two-plasmid expression system, (B) the plasmid based on the pET29b vector coding gene for NMT used in the two-plasmid expression system, (C) the plasmid carrying both MAPPHis and NMT genes constructed on the basis of the pET19b and pET11b vectors used in the single-plasmid system.

Figure 2

Coomassie stained SDS-PAGE gel illustrating the production of myrMAPPHis and cell lysis. Odd lanes show proteins produced from the two-plasmid system, even lanes show proteins produced in the single-plasmid system. Lanes: (1) broad range SDS-PAGE standard (Bio-Rad), (2, 3) cells before induction, (4, 5) cells 4h after induction, (6, 7) supernatant after cell lysis, (8, 9) pellet after cell lysis. MAPPHis is the large band with Mw 14.5 kDa, lanes 6–9 also contain lysozyme, which has similar mobility as MAPPHis. NMT has molecular weight of 55 kDa, but it can not be distinguished from other bacterial proteins.

Figure 3

Coomassie stained SDS-PAGE gel illustrating purification of myrMAPPHis produced using the single-plasmid system. Lanes 3–5 show purification of the first aliquot directly eluted by imidazole buffer and lanes 6–9 show purification of the second aliquot which was cleaved on Ni-NTA and then eluted by imidazole. Lanes: (1) broad range SDS-PAGE standard (Bio-Rad), (2) flow-through after binding (14 kDa band is lysozyme), (3) protein eluted from column using imidazole buffer, (4) sample of Ni-NTA agarose after elution, (5) eluate (the same as in lane 3) cleaved by Pr13 for 1 hour, (6) protein cleaved from Ni-NTA by Pr13, (7) protein eluted from column using imidazole buffer after cleavage, (8) sample of Ni-NTA agarose after elution, (9) eluate (the same as in lane 7) cleaved by Pr13 for 1 hour

Figure 4

Coomassie stained SDS-PAGE gel illustrating Ni-NTA purification of myrMAPPHis produced using the two-plasmid system. Lanes 3–5: purification of the first aliquot directly eluted by imidazole buffer and lanes 6–9: purification of the second aliquot, which was cleaved on Ni-NTA and then eluted by imidazole. Lanes: (1) broad range SDS-PAGE standard (Bio-Rad), (2) flow-through after binding (14 kDa band is lysozyme), (3) proteins eluted by imidazole buffer, (4) sample of Ni-NTA agarose after elution, (5) eluate (the same as in lane 3) cleaved by Pr13 for 1 hour, (6) protein cleaved from Ni-NTA by Pr13, (7) protein eluted from column using imidazole buffer after cleaving, (8) sample of Ni-NTA agarose after elution, (9) eluate (the same as in lane 7) cleaved by Pr13 for 1 hour

Figure 5

MALDI-TOF MS spectrum of purified myrMAPPHis obtained from the single-plasmid system (same sample as in Fig. 3 lane 7). The m/z ratio of larger peak corresponds to Mw of non-myristoylated MAPPHis.

Figure 6

MALDI-TOF MS spectrum of myrMAPPHis obtained from the two-plasmid system without cleavage by Pr13 on Ni-NTA (same sample as in Fig. 4 lane 3). Mw The m/z ratio of larger peak corresponds to Mw of myristoylated MAPPHis. Peak with m/z ratio of 15116 is an adduct of myrMAPPHis and MS matrix.

Figure 7

MALDI-TOF MS spectrum of purified myrMAPPHis obtained from the two-plasmid system (the same sample as in Fig. 4 lane 7). The m/z ratio of larger peak corresponds to Mw of myristoylated MAPPHis. Peak with m/z ratio of 15109 is again an adduct of myrMAPPHis and MS matrix.

Figure 8

Overlay of region of ¹H-¹³C HSQC spectra, measured on myrMAPPHis (red) and MAPPHis (blue), showing signals of gamma methyl groups of isoleucines 86 and 90 and valines 38 and 59, respectively.

Figure 9

Overlay of ¹H-¹⁵N HSQC spectra measured on MAPPHis (red) and MA (blue). Signals with different chemical shifts are located at the C-terminus of MA.