Chapter 1
Production of Multi-subunit Membrane Protein Complexes
Burak V. Kabasakal, Qiyang Jiang, and Christiane Schaffitzel
Abstract
Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of membrane proteins and their complexes is of
key importance, but the progress in this area is slow because of the difficulties to produce them in sufficient
quality and quantity. Overexpression of membrane proteins is often restricted by the limited capability
of translocation systems to integrate proteins into the membrane and to fold them properly. Purification of
membrane proteins requires their isolation from the membrane, which is a further challenge. The choice of
expression system, detergents, and purification tags is therefore an important decision. Here, we present a
protocol for expression in bacteria and isolation of a seven-subunit membrane protein complex, the
bacterial holo-translocon, which can serve as a starting point for the production of other membrane protein
complexes for structural and functional studies.
Key words Membrane protein complex, Expression in bacteria, Endogenous host, Complex purification, Translocon
1 Introduction
1.1 Aim of the Study
Membrane proteins represent more than 25% of the proteome of all
cells. They mediate the cell’s interaction with its environment, i.e.,
transmission of signals, cell adhesion, and transport across membranes. Membrane proteins are of prime pharmaceutical interest as
they constitute ~50% of known potential drug targets. In addition
to this, membrane proteins are the natural entry and/or anchoring
points for infectious agents. Dysfunctional membrane proteins are
the basis of many disorders such as cystic fibrosis and Alzheimer’s.
Understanding the function and molecular structure of this class of
proteins is of key interest.
As in the cytosol, the majority of proteins in the membrane
occur in and function in complexes. For instance in budding yeast,
membrane proteins were found to interact on average with two to
three interaction partners [1]. Due to their association with cellular
membranes, expression and purification of membrane proteins and
their complexes are often a challenging task. Progress is often
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3
Production of Multi-subunit Membrane Protein Complexes
Burak V. Kabasakal, Qiyang Jiang, and Christiane Schaffitzel
Abstract
Membrane proteins constitute an important class of proteins for medical, pharmaceutical, and biotechnological reasons. Understanding the structure and function of membrane proteins and their complexes is of
key importance, but the progress in this area is slow because of the difficulties to produce them in sufficient
quality and quantity. Overexpression of membrane proteins is often restricted by the limited capability
of translocation systems to integrate proteins into the membrane and to fold them properly. Purification of
membrane proteins requires their isolation from the membrane, which is a further challenge. The choice of
expression system, detergents, and purification tags is therefore an important decision. Here, we present a
protocol for expression in bacteria and isolation of a seven-subunit membrane protein complex, the
bacterial holo-translocon, which can serve as a starting point for the production of other membrane protein
complexes for structural and functional studies.
Key words Membrane protein complex, Expression in bacteria, Endogenous host, Complex purification, Translocon
1 Introduction
1.1 Aim of the Study
Membrane proteins represent more than 25% of the proteome of all
cells. They mediate the cell’s interaction with its environment, i.e.,
transmission of signals, cell adhesion, and transport across membranes. Membrane proteins are of prime pharmaceutical interest as
they constitute ~50% of known potential drug targets. In addition
to this, membrane proteins are the natural entry and/or anchoring
points for infectious agents. Dysfunctional membrane proteins are
the basis of many disorders such as cystic fibrosis and Alzheimer’s.
Understanding the function and molecular structure of this class of
proteins is of key interest.
As in the cytosol, the majority of proteins in the membrane
occur in and function in complexes. For instance in budding yeast,
membrane proteins were found to interact on average with two to
three interaction partners [1]. Due to their association with cellular
membranes, expression and purification of membrane proteins and
their complexes are often a challenging task. Progress is often
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3