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

Abstract

Mistic, a bacterial membrane-associating protein family, uniquely found in Bacillus species. It enhances expression of eukaryotic membrane proteins at the bacterial membrane. Mistic from B. subtilis (M110), expresses at the E. coli membrane, however its shorter orthologs have been recently shown to be mainly cytoplasmic with varying membrane affinities. Based on that, we hypfothesized that the expression level of membrane proteins fused to Mistic is correlated with the degree of membrane association of the particular Mistic protein. We compared expression levels by various Mistic proteins as fusion partners for the Aplysia californica Kv1.1 (aKv1.1) channel as a cargo membrane protein. Mistic from B. atrophaeus (M4), which has the highest membrane association among the shorter orthologs, enhanced expression of the transmembrane domain of aKv1.1 to the highest extent. In contrast, M1, which consists of the 84 C-terminal amino acids of M110 is the most soluble protein and showed the least capacity to express the channel. A chimeric Mistic, constructed with the first α-helix (H1) of M110 N-terminally fused to M4, did not increase the level of expression of aKv1.1 beyond those of either the M110 or the M4 fusions. The channel fused to M110, M4 or the aforementioned H1-M4 chimera, expresses in the highest quantity and quality among Mistic proteins, providing suitable sample for structural studies. Our data support the concept that expression levels of 'Misticated' membrane proteins are related to the independent chaperoning character of Mistic via direct membrane association, rather than related to specific sequence-dependent interaction with the E. coli translocon machinery. KeywordsMistic; membrane proteins; potassium channel; overexpression 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 Ca 2+ 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, © 2009 Elsevier Inc. All rights reserved.Address correspondence to: Senyon Choe <choe@salk.edu>. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. there is accordingly an increasing demand for more robust techniques of expressin...

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…J. 2012, 7, 620-634 www.biotechnology-journal.com nism of action remains unclear; however, in some cases the use of Mistic and its orthologs have significantly increased levels of eukaryotic integral membrane protein expression in E. coli [73,74,76].…”

Strategies of Bacterial Over Expression of Membrane Transporters Relevant in Human Health: The Successful Case of the Three Members of OCTN Subfamily

“…Besides the most known 6, or longer, His tag, which is used essentially for purification [42], GST, MBP, NusA, Trx, and SUMO [43, 44] are potentially useful for improving solubility or even expression yield. For the expression of membrane proteins a promising approach might be the fusion with Mistic [45, 46], a small E. coli outer membrane protein, which can promote the insertion of the fusion peptide in the E. coli membrane to help the folding process. Chaperones co-expression has also been attempted to reduce formation of insoluble aggregates responsible for protein inactivation and difficult refolding.…”

MstX and a Putative Potassium Channel Facilitate Biofilm Formation in Bacillus subtilis

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