Technical Highlight - January 2010
Short description: A refinement of the SPP method eliminates lengthy purification steps in the pursuit of membrane proteins suitable for structural studies.
Purifying membrane proteins for structural studies is usually a time-consuming and costly task. Now Masayori Inouye and colleagues from PSI NESG and NYCOMPS have adjusted their single protein production (SPP) technique to work with integral membrane proteins. Here are five reasons to use their method.
1. No purification required
Thanks to the highly enriched expression of the desired protein, a purification step is no longer needed, although a one-step purification on an affinity chromatography column improves the quality of the NMR spectra by eliminating unwanted phospholipids.
The condensed SPP (cSPP) system relies on producing the desired protein in bacteria expressing MazF, an mRNA interferase that removes all mRNAs containing the sequence. For example, the team substituted all the ACA sequences in the gene encoding the inner membrane protein YaiZ. They then induced expression of the modified YaiZ and MazF in Escherichia coli, separated the inner and outer membrane fractions, and found that YaiZ was the most abundant protein in the inner membrane. YaiZ was the only protein that was labeled with isotopes so that NMR studies on this membrane protein can be done without purification.
2. Saves money
Cells predominantly expressing YaiZ were resuspended in a much smaller volume of minimal medium for isotope labeling with 15NH4Cl than is normally used. Reducing the amount of suspension medium hugely reduces the cost of isotope labeling, with savings of up to 99%.
3. Easy protein deuteration
In this system, 2H2O does not inhibit protein production, unlike its effects in many other protein expression set-ups, and so it is possible to produce high-quality deuterium-enriched samples of soluble or membrane proteins. For small, soluble proteins, this enables reliable automated resonance assignments and the generation of three-dimensional structures.
4. Ideal for solid-state NMR
Solid-state NMR is one of the few non-invasive methods that can be used for membrane protein studies. The protein-enriched membrane fraction produced by cSPP is ideal for solid-state NMR studies.
5. Easy detergent screening
This cSPP system with its selective isotope enrichment of the target protein provides a straightforward way to screen for detergent buffer conditions without having to go through the whole purification process. The team performed high-throughput screening using a microcoil NMR probe to produce 1D NMR spectra.
The cSPP system shows that it is possible to obtain NMR data for membrane proteins without purification other than a simple membrane extraction. This method is quite general and is likely to be applicable to many other NMR-based studies.
L. Mao, Y. Tang, S. T. Vaiphei, T. Shimazu, S.-G. Kim et al. Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system.
J. Struct. Funct. Genomics (2009). doi:10.1007/s10969-009-9072-0