An optimized Npro-based method for the expression and purification of intrinsically disordered proteins for an NMR study

Abstract

Intrinsically disordered proteins (IDPs) are an emerging concept. IDPs have high flexibility in their polypeptide chains, lacking a stable 3-dimensional structure. Because of the difficulty in performing X-ray crystallography for IDPs, nuclear magnetic resonance (NMR) spectroscopy is the first choice for atomic-level investigation of their nature. Given that isotopically labeled IDP samples are necessary for NMR study, a robust and cost-effective protocol for bacterial expression and purification of IDP is also needed. We employed the N^pro (EDDIE)-autoprotease fusion protein system. Although IDPs are believed to be readily degraded by endogenous proteases when expressed in Escherichia coli, N^pro-fused IDPs showed excellent resistance to degradation. Seven IDPs of uncharacterized function sampled from the human genome as well as 3 constructs from IDP regions derived from human FancM and Thermococcus kodakarensis Hef were prepared. We improved the protocol of refolding of N^pro (EDDIE) to use dialysis, which is convenient for subsequent purification using reversed-phase (RP) HPLC. The method is robust and widely applicable to any IDP sample, promoting the acquisition of experimental data for IDPs in a high-throughput manner.

Figure 1.

Examples from the expression–purification step of an N^pro (EDDIE)-IDP(H1) fusion protein analyzed by SDS-PAGE (15% gel). (A) sup or ppt indicates soluble or pellet fraction of sonicated extract from IPTG-induced cells, respectively (lanes 1, 2). (B) Ni²⁺ affinity column purified N^pro (EDDIE)-IDP(H1) in denatured condition (lanes 3–7) with a given imidazole concentration. (C) dialysis and autocleavage of N^pro (EDDIE)-IDP(H1). MW markers (lane M), before refolding (lane 8), after the first dialysis step (lane 9), after an additional dialysis step (lane 10), supernatant after without salt/DTT buffer dialysis (lane 11), and pellet of the same dialysis (lane 12). (D) A chart of RP-HPLC purification of IDP(H1). Inset is a acetonitrile gradient profile. In (C) and (D), open circle and closed diamond represent N^pro and IDP(H1), respectively. In (D), asterisk indicates a partially degraded IDP(H1).

Figure 2.

MALDI-TOF mass spectra and HSQC/SOFAST-HMQC spectra of purified IDPs. MALDI-TOF mass spectra (A–F) and NMR spectra (G–L). In the NMR spectra, the SOFAST-HMQC spectrum measured in the buffer with 0 M NaCl is shown in cyan, whereas the HSQC spectrum in 0.5 M NaCl is shown in red. (A, G) IDP(B3), (B, H) IDP(B4), (C, I) IDP(C1), (D, J) IDP(D10), (E, K) IDP(E1), (F, L) “partial”-IDP(C9).