Abstract
Lack of high-quality antibodies against transmembrane proteins is a widely recognized hindrance in biomedical and cell biological research. Here we present a robust pipeline for the generation of polyclonal antibodies employing full-length membrane proteins as immunogens to overcome this “antibody bottleneck”. We express transmembrane proteins fused to a MISTIC fragment that enhances expression of eukaryotic membrane proteins in E. coli. Purified membrane proteins are used as immunogen for rabbit injection employing standard immunizing protocols. The raised antibodies against membrane proteins of the endoplasmic reticulum and the nuclear envelope, which we use as test cases, function in a wide range of applications and are superior to ones produced against soluble domains as immunogens.
Highlights
Human cells have a wide variety of integral membrane proteins, which comprise approximately 30% of proteins encoded by the genome[1,2]
A 110 aa membrane associated protein from Bacillus subtiliis, MISTIC, has been shown to enhance the expression levels of foreign integral membrane proteins in E. coli when generated as fusion proteins[12,13,14]
We present a simple workflow using MISTIC-fusion proteins for high-yield expression of eukaryotic transmembrane proteins in E. coli., which are used for efficient polyclonal antibody production
Summary
Human cells have a wide variety of integral membrane proteins, which comprise approximately 30% of proteins encoded by the genome[1,2]. Expression in yeast, plants, insect and mammalian cells as well as cell free systems have been employed for generation and purification of integral membrane proteins[6,7,8,9,10] These techniques involve relative high costs and the success rate is often unpredictable. Bacteria, most importantly Escherichia coli, are the most widely used hosts when attempting production of membrane proteins[11] This is despite the fact that integral membrane proteins often express in relative low yields as compared to soluble proteins or are found in inclusion bodies which requires denaturing purification protocols. Unusual for a bacterial membrane protein MISTIC is highly hydrophilic and lacks a detectable signal sequence[14] It may avoid the bacteria’s translocon machinery to integrate into the bacteria membrane in a Sec independent manner. The obtained antisera were compared to antisera generated against the soluble domains of the respective proteins in biochemical applications including western blotting, immunoprecipitation and immunofluorescence and were found to be superior in all aspects
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