Bacterial expression of a eukaryotic membrane protein in fusion to various Mistic orthologs.

Integral membrane proteins (IMPs) constitute approximately a quarter of any eukaryotic or prokaryotic proteome. Irrespective of their prevalence, structural characterization of IMPs has been very limited due to the challenges arising from the hydrophobic nature of their transmembrane (TM) domains. Although some eukaryotic IMPs, such as rhodopsin and Ca2+ ATPase, are found in high amounts in native biological membranes, most membrane proteins do not normally exist in such abundance. With the advent of the vast amount of genomic information and major advances in high-throughput structural biology techniques, there is accordingly an increasing demand for more robust techniques of expressing of IMPs to advance structural knowledge about eukaryotic IMPs. The first major obstacle for structural studies of IMPs is the production of large amounts of properly folded protein. Unlike soluble proteins, highly hydrophobic membrane proteins require auxiliary proteins to prevent their aggregation during the protein folding process [4, 5]. It is thus likely that production of eukaryotic IMPs in bacteria is limited compared to that of soluble proteins, due to the additional complexity involved in the biogenesis of IMPs, coordinated by the ribosome and the SecYEG translocon assisted by chaperones such as SecB and the ATPase motor SecA, some of which may be needed to be co-overexpressed and/or impose additional specificity [6–10]. The next obstacle is to isolate the IMP from the membrane for further purification, which typically requires screening for the proper detergent to allow the formation of a stable mono-dispersed protein-detergent-complex (PDC). Finally, once a pure PDC is obtained (by purification techniques similar to those applied for soluble proteins), an extensive search for a detergent or a mixture of detergents with or without lipids is often needed to promote successful crystallization of the IMP in the PDC. Overcoming the above barriers for successful crystallization of IMPs requires much larger protein quantities on average, although ironically their expression yields are generally much lower than those for soluble proteins. This difference entails the challenge in structural biology of IMPs, and highlights the importance for the development of high-yield expression systems for IMPs. Mistic is a membrane-associated protein originally discovered in Bacillus subtilis [11]. It was shown to enhance expression levels of a large number of foreign IMPs at the membrane of E. coli, when used as a fusion partner linked to the N-termini of cargo proteins [11–15]. Interestingly, Mistic lacks identified signal sequence and its overexpression, whether alone or in fusion to other IMPs, apparently lacks the toxicity issues associated with overloading the protein translation machinery, and thus is likely to bypass the E. coli translocon complex [11]. Other Mistic proteins have been identified in Bacillus species only, and they are 26 amino acids shorter than the Mistic originally identified from B. subtilis [12], referred to as M110. The discovery of shorter Mistic orthologs provided new opportunities for optimizing expression of IMPs, given that they were all shown to serve as fusion partners for expression of IMPs at the bacterial membrane [12]. Surprisingly, it was recently shown that, unlike M110, the shorter Mistic proteins are soluble in the absence of detergents and they are mainly cytoplasmic, but show significantly varying membrane association propensities [16]. In accordance with previous suggestion on the independent ability of Mistic to facilitate membrane expression of IMPs, that study indicates that isolated Mistic proteins may spontaneously insert into the membrane. Based on that, we hypothesized that the degree of membrane association displayed by the individual Mistic proteins [16], is correlated to their ability to enhance expression of IMPs at the bacterial membrane. Despite many efforts, eukaryotic voltage-gated potassium channels have not been successfully expressed in bacteria unless fused to Mistic. To test our hypothesis, we have chosen the Aplysia californica Kv1.1 (aKv1.1) channel, which showed no basal level of expression in E. coli, as a cargo IMP for the comparison of its expression by various Mistic fusion partners. We report on the capacity of various Mistic orthologs in assisting expression of aKv1.1 in the bacterial membrane, which enabled us to achieve large quantities of pure and stable ‘Misicated’ eukaryotic IMP. Finally we discuss Mistic’s mechanistic roles in chaperoning the insertion of IMPs fused to its C-terminus into the lipid-bilayer membrane.