Membrane proteins are notoriously difficult to express. Moreover, expressing individual subunits from large membrane protein complexes poses a particular challenge. Several issues need to be addressed while designing and developing protocols:

• Is the protein expressed?
• Is the protein stable post-expression?
• Can the protein be detected, and thus expression conditions optimized?
• Can the protein be successfully isolated for functional studies?

Nowadays, most of these challenges can easily be solved using advanced techniques or sometimes even with brute force high-throughput testing. However, this was not the case in the late 2000s.

Initial Attempts and Setbacks

Traditionally, to check the expression of a new protein, a Western blot can be used to assess the production levels. Unfortunately, our initial efforts to produce His-tagged subunits of respiratory chain complex I in E. coli were met with low yields and detection difficulties. Membrane proteins, being poorly antigenic, required the creation of a specific antibody from a synthetic peptide, which unfortunately did not react well with the native proteins. We simply could not tell if the protein was not detected or was not produced at all. This led us to consider fusing the polypeptides with a protein domain that was colorful, small, and non-toxic to E. coli cells.

Innovation: Cytochrome c Fusion

While GFP may be the obvious choice for a tag, it does not fold correctly in the periplasm. We therefore chose to use the C-terminal cytochrome c550 domain from Bacillus subtilis as a fusion partner due to its small size and periplasmic haem-binding domain, giving it a distinctive bright red color. All other advantages aside, it is simply much easier to work with a protein that you can see.

Outcomes and Advantages

The red color of cytochrome c fusion proteins allowed for quick assessment of expression levels in whole cells, as well as following the purification progress. Proteins could be seen at concentrations as low as 0.1 mg/ml, significantly speeding up handling and reducing the need for frequent absorbance measurements and SDS-PAGE.

The heme group in cytochrome c, bound covalently via thioether bonds, remained intact even after protein denaturation, facilitating detection through peroxidase activity staining or chemiluminescence assays. Often, the band could even be seen on unstained SDS-PAGE gels.

Being able to readily detect and quantify the expression levels, we could easily optimize the expression protocols, resulting in high yields of high-quality protein. Additionally, the compact cytochrome c tag domain improved the stability of the expressed proteins, as the native proteases could not digest the proteins. Lastly, the His-tag at the C-terminus of the cytochrome c domain was exposed and allowed for easy purification to very high purity in a one-step metal affinity purification protocol.

Publications:

1. E. Sperling, K. Górecki, T. Drakenberg, C. Hägerhäll, Functional Differentiation of Antiporter-Like Polypeptides in Complex I; a Site-Directed Mutagenesis Study of Residues Conserved in MrpA and NuoL but Not in MrpD, NuoM, and NuoN, (2016), PLOS ONE 11, 7. Read
2. K. Górecki, C. Hägerhäll, T. Drakenberg, The Na⁺ transport in Gram-positive bacteria defect in the Mrp antiporter complex measured with ²³Na-NMR, (2014) Analytical Biochemistry 445: 80-86. Read (open access at PubMed)
3. V. K. Moparthi, B. Kumar, Y. Al-Eryani, E. Sperling, K. Górecki, T. Drakenberg, C. Hägerhäll, Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I, (2014), BBA – Bioenergetics 1837: 178-185. Read
4. E. Virzintiene, V. K. Moparthi, Y. Al-Eryani, L. T. Shumbe, K. Górecki, C. Hägerhäll, Structure and function of the C-terminal end of MrpA in the Bacillus subtilis Mrp-antiporter complex – the evolutionary progenitor of the long, membrane parallel helix in Complex I. (2013) FEBS Letters 587 (20): 3341-3347. Read
5. T. Gustavsson, M. Trane, V. K. Moparthi, E. Miklovyte, L. Moparthi, K. Górecki, T. Leiding, S. Petersson Årsköld, C. Hägerhäll, A cytochrome c-fusion protein domain for convenient detection, quantification and enhanced production of membrane proteins in Escherichia coli – expression and characterisation of cytochrome-tagged complex I subunits, (2010) Protein Science 19: 1445-1460. Read