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Step 2: Growth of L. lactis, Expression of the Target Protein and Preparation of Membrane Vesicles

Step 2: Growth of L. lactis, Expression of the Target Protein and Preparation of Membrane Vesicles

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Membrane Protein Expression in Lactococcus lactis



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5. Centrifuge (6000 Â g, 10 min, 4 °C) and discard the supernatant.

Resuspend the pellet in TBS and centrifuge (6000 Â g, 10 min, 4 °C).

Resuspend the pellet in TBS, aliquot the sample into 15-mL falcon

tubes, and centrifuge (4000 Â g, 10 min, 4 °C). Discard the supernatant,

snap-freeze thoroughly in liquid nitrogen, and store at À80 °C

6. If using whole cells for functional assays, it is imperative that the frozen

cells are “snap-thawed” to prevent cell lysis. Add 2 mL TBS to the frozen

pellet, and resuspend by gentle pipetting. To aid thawing, place the falcon tube in a beaker of warm water during resuspension; place the

thawed cells on ice

Isolation of L. lactis membrane vesicles

1. Thaw the frozen cells, and lyse by mechanical disruption with one pass at

30 kpsi through a cell disruptor. We use a Constant Systems Cell

disrupter.

2. Centrifuge the disrupted sample at low speed (11,000 Â g, 15 min, 4 °C) to

remove whole cells and debris; decant the supernatant into ultracentrifuge

tubes, and centrifuge (200,000 Â g, 1 h, 4 °C) to collect the membranes.

3. Resuspend the pellet in TBS and centrifuge (200,000 Â g, 1 h, 4 °C) to

wash the membranes.

4. Resuspend the pellet in TBS and homogenize; determine the concentration using the BCA assay, and adjust the concentration to 5 mg/mL;

snap-freeze and store in liquid nitrogen.

Analysis

Determine expression using SDS-PAGE and Western blot analysis

according to standard protocols. For highly expressed proteins, a band

may be seen on a Coomassie-stained SDS-PAGE gel; for lowerexpressed proteins, Western blot analysis will be necessary to confirm

expression.



6.2 Tips

– After the addition of nisin A, swirl immediately to promote global induction and to prevent localized lysis, as nisin A inserts into the membrane to

form pores.

– Using the correct procedures (as detailed above), whole cells can be frozen and stored at À80 °C without adverse effects.

– The one-shot disruption system of Constant Systems is the most suitable

system to disrupt the lactococcal cell wall as the yield of crude membranes

improves more than fivefold when compared to that obtained by lysozyme

and French press treatment (Frelet-Barrand, Boutigny, Kunji, et al., 2010).



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ACKNOWLEDGMENT

This research was supported by the Medical Research Council.



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pone.0022981, ARTN e22981.



CHAPTER FIVE



An Unconventional Anaerobic

Membrane Protein Production

System Based on Wolinella

succinogenes

Michael Lafontaine, C. Roy D. Lancaster1

Department of Structural Biology, Institute of Biophysics and Center of Human and Molecular Biology

(ZHMB), Saarland University, Homburg, Germany

1

Corresponding author: e-mail address: roy.lancaster@structural-biology.eu



Contents

1. Introduction

1.1 pΔfrdCAB

1.2 pFrdcat2

1.3 Construction of W. succinogenes strains

2. Methods

2.1 Materials

2.2 Preparation of growth media

2.3 Cultivation of W. succinogenes

2.4 Generation of the expression vectors

2.5 Transformation and genomic integration

2.6 Stock culture

2.7 Protein production in W. succinogenes

3. Purification of Proteins Expressed in W. succinogenes

3.1 Material

3.2 Buffers

3.3 Cell lysis procedure for the extraction of a membrane protein

3.4 Cell lysis procedure for the extraction of a periplasmic protein

3.5 Anion exchange chromatography

3.6 Size-exclusion chromatography

3.7 Determination of protein concentration

3.8 Functional characterization

3.9 Reconstitution of enzymes in proteoliposomes

3.10 Enzymic assays

Acknowledgments

References



Methods in Enzymology, Volume 556

ISSN 0076-6879

http://dx.doi.org/10.1016/bs.mie.2014.12.026



#



2015 Elsevier Inc.

All rights reserved.



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Michael Lafontaine and C. Roy D. Lancaster



Abstract

In cases where membrane protein production attempts in more conventional

Escherichia coli-based systems have failed, a solution is to resort to a system based

on the nonpathogenic epsilon-proteobacterium Wolinella succinogenes. This approach

has been demonstrated to be successful for structural and mechanistic analyses not

only for homologous production of W. succinogenes membrane proteins but also for

the heterologous production of membrane protein complexes from the human pathogens Helicobacter pylori and Campylobacter jejuni. The procedure to establish a system

for the production of native and variant enzymes in W. succinogenes is presented in

detail for the examples of the quinol:fumarate reductase and the SdhABE complexes

of W. succinogenes. Subsequently, further projects using W. succinogenes as expression

host are covered.



1. INTRODUCTION

A prerequisite for the understanding of the mechanism of action of

membrane proteins at an atomic level is the availability of accurately determined three-dimensional structures. The by far most successful technique in

the determination of atomic models of membrane protein structure is X-ray

crystallography ( Jaskolski & Wlodawer, 2014; Schmahl & Steurer, 2012;

Wilkins, 2013). This method requires the crystallization of the membrane

protein of interest (Michel, 1990; M€

uller & Lancaster, 2013; Newby

et al., 2009), which in turn requires its production and purification in milligram quantities and monodisperse quality (Ostermeier & Michel, 1997).

Although a number of well-established membrane protein production systems, based on bacteria (Geertsma & Poolman, 2010; Makrides, 1996;

Miroux & Walker, 1996), yeast (Cereghino & Cregg, 2000; Cregg,

Cereghino, Shi, & Higgins, 2000), or insect cells ( Jasti, Furukawa,

Gonzales, & Gouaux, 2007), are available, (in particular heterologous)

expression can fail for a variety of reasons. An alternative system for membrane proteins, where Escherichia coli-based production failed, is based on the

epsilon-proteobacterium Wolinella succinogenes and is presented here. It has

been demonstrated to be successful for both homologous (Herzog et al.,

2012; Juhnke, Hiltscher, Nasiri, Schwalbe, & Lancaster, 2009; Lancaster,

Gross, & Simon, 2001; Lancaster et al., 2000, 2005) and heterologous

(Mileni et al., 2006) membrane protein production, crystallization, and

membrane protein structure determination (Lancaster et al., 2000, 2001;

Lancaster, Kr€

oger, Auer, & Michel, 1999; Lancaster et al., 2005; Madej,



Wolinella succinogenes Membrane Protein Production



101



Nasiri, Hilgendorff, Schwalbe, & Lancaster, 2006). The system is based on

earlier work by the laboratory of the late Kr€

oger (K€

ortner, Lauterbach,

Tripier, Unden, & Kr€

oger, 1990; Kr€

oger et al., 2002; Lauterbach,

K€

ortner, Albracht, Unden, & Kr€

oger, 1990; Simon, Gross, Ringel,

Schmidt, & Kr€

oger, 1998). Of central importance is the W. succinogenes

quinol:fumarate reductase (QFR) deletion mutant (ΔfrdCAB) first generated by Simon et al. (1998). In the following, the procedure to establish a

system for the production of native and variant enzymes in

W. succinogenes is presented for the example of QFR variants and the

SdhABE complex of W. succinogenes. In addition, further projects using

W. succinogenes as expression host are covered.



1.1 pΔfrdCAB

Deletion of genomic frdCAB was described by Simon et al. (1998). In brief, a

kanamycin deletion cassette of pUC4K was inserted in the pBR322 vector

flanked by two DNA sequences corresponding to genomic regions upstream

and downstream of the frdCAB operon. Ligation reactions and subsequent

plasmid propagation were performed in the E. coli XL-1 blue strain. Transformation with this vector and subsequent selection with kanamycin yielded

recombinant clones of W. succinogenes ΔfrdCAB mutant, where the genomic

frdCAB operon was replaced with the kanamycin cassette via a double

homologous recombination events. These mutants are not able to grow

on fumarate but still on nitrate minimal medium.



1.2 pFrdcat2

The expression of different frdCAB variants is achieved by transforming the

ΔfrdCAB mutant with the pFrdcat2 plasmid (Simon et al., 1998). This plasmid (Fig. 1A) is a derivative of the pFrd vector where the frdC2 gene and the

kanamycin resistance gene (kan) are mostly deleted but it contains the chloramphenicol resistance gene (catGC) of the pDF4 vector. The sequence of this

plasmid was determined by Juhnke et al. (2009) and is deposited in the

EMBL nucleotide sequence database (accession no. AM909725). Ligation

reactions and subsequent plasmid propagation were performed in E. coli

XL-1 blue strain. Transformation with pFrdcat2 complements ΔfrdCAB

as the plasmid integrates into the genome via a single recombination event

between the sequence upstream of frdC in the vector and the corresponding

genomic region. The resulting complemented deletion mutant, also referred



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Michael Lafontaine and C. Roy D. Lancaster



Figure 1 Use of the pFrdcat2 vector for the generation of W. succinogenes QFR variants.

(A) Construction of the W. succinogenes △frdCAB mutant. A double homologous recombination event between flanking regions present in the p△frdCAB vector and the

genome of W. succinogenes leads to the replacement of the genomic frdCAB locus by

a kanamycin deletion cassette (Kan). (B) Integration of the pFrdcat2 vector via a single

homologous recombination event into the genome of the W. succinogenes △frdCAB

mutant. The recombination event takes place between a region upstream of the frdC

gene present in the pFrdcat2 vector and the genome. Simplified representation adapted

from Simon et al. (1998).



Wolinella succinogenes Membrane Protein Production



103



as K4, shows in terms of doubling time and fumarate reductase activity similar properties like the wild-type strain (Simon et al., 1998).



1.3 Construction of W. succinogenes strains

The generation of recombinant W. succinogenes strains comprises two important steps. The first step involves the deletion of the genomic locus of a certain gene via a double homologous recombination event. In case of the

QFR of W. succinogenes, the genomic frdCAB operon which encodes for

the QFR subunits is replaced by a deletion cassette of the pΔfrdCAB. This

deletion cassette contains a kanamycin resistance gene of pUC4K flanked by

two DNA sequences corresponding to genomic regions upstream and

downstream of the frdCAB operon. The flanking regions were synthesized

by PCR from the pFrd and pPur adding a EcoRI and BamHI restriction site

in case of the upstream fragment and BamHI and SalI for the downstream

region (Simon et al., 1998). Both fragments were cloned in the pBR322

vector before inserting the kanamycin resistance gene via BamHI. Transformation of W. succinogenes with this vector and subsequent selection with

kanamycin yielded recombinant clones of W. succinogenes ΔfrdCAB mutant

(also referred as the deletion mutant), where the genomic frdCAB operon

was replaced by the kanamycin cassette. These mutants are not able to grow

on fumarate but still on nitrate minimal medium.

The next step complements this mutation by integrating the pFrdcat2

vector via a single recombinant event. The pFrdcat2 vector (Fig. 1B), a

modified pFrd vector, was constructed by deleting most of the kanamycin

resistance gene and inserting the chloramphenicol resistance gene from

pDF4a. A subsequent digestion of the vector with ClaI and SalI followed

by ligation resulted in the frdCAB operon lacking the C2 open reading

frame. The sequence of this plasmid is deposited in the EMBL nucleotide

sequence database (accession no. AM909725). The complete pFrdcat2 plasmid is integrated into the genome via a single recombination event between

the 0.7-kb fragment upstream of the frdC open reading frame in vector and

genome of the deletion mutant (Fig. 2). Selection on media containing

kanamycin and chloramphenicol (25 and 12.5 μg/mL, respectively) yielded

the complemented deletion mutant K4 that shows wild-type properties in

terms of doubling time, growth yield, and specific activity for fumarate respiration (Simon et al., 1998).

For the heterologous expression of membrane protein complexes, as performed by Mileni et al. (2006), this system is still applicable. Since the QFR



Figure 2 General workflow to generate recombinant W. succinogenes strains. After generation of the expression vectors (1) and plasmid production in E. coli XL-1 blue cells (2),

transformation of W. succinogenes wild-type or W. succinogenes △frdCAB mutants with

the expression vectors leads to their integration into the genome (3). Selection procedure and PCR screening for genomic integration of the vector (4) yield stably transformed W. succinogenes strains that can be used for subsequent experiments (5).



Wolinella succinogenes Membrane Protein Production



105



enzymes of the epsilon-proteobacterial human pathogens Helicobacter pylori

(Ge et al., 2000) and Campylobacter jejuni (Weingarten, Taveirne, &

Olson, 2009) have been shown to be essential for colonization of the host

organism, these QFR enzymes are considered to be promising drug targets.

After generating the deletion mutant as previously described (Simon et al.,

1998), the cells were transformed with the pCatCj4 and pCatHpG8 plasmids. These derivatives of the pFrdcat2 vectors contain the frdCAB locus

of C. jejuni and H. pylori, respectively, instead of the genuine W. succinogenes

frdCAB locus. Nevertheless, the frdCAB locus is still under the control of the

strong frd promoter. The derivative vectors were constructed by amplifying

an frdCAB lacking fragment of pFrdcat2 and the respective frdCAB loci with

primers containing a ClaI and an AvrII restriction site at their 50 ends. Transformants (W. succinogenes CjM11 and HpGM31) were able to grow on kanamycin and chloramphenicol and produced functional heterologous QFR

enzymes with expression levels comparable to the homologous wild type

(Mileni et al., 2006).

However, this system is not only applicable to QFRs demonstrated by

the work of Kern, Scheithauer, Kranz, and Simon (2010) or Juhnke et al.

(2009). The latter used the genetic system to produce a nonclassical succinate:quinone oxidoreductase (SQOR) (E-type) of W. succinogenes. This

enzyme, classified as an E-type SQOR (Hederstedt, 1999; Lancaster,

2002a), has not been produced yet under any tested growth condition.

The SdhABE operon encodes the three distinct subunits of the enzyme

which is composed of two hydrophilic subunits (SdhA and SdhB) and a

membrane anchor (SdhE) which is predicted to be membrane associated

via amphipathic helices than a transmembrane domain. Furthermore, the

SdhA subunit contains a N-terminal 40-amino acid-long extension harboring a twin-arginine motif that predetermines the protein to be exported via

the tat pathway (Palmer, Sargent, & Berks, 2005).

As the cloning of the complete SdhABE operon failed, only the gene

coding for sdhA subunit was amplified from genomic DNA and cloned

via SacII and NotI in a pFrdcat2 fragment lacking the frdCAB operon but

still containing the intact frd promoter (referred as pSdhA). Transformation

of the W. succinogenes ΔfrdCAB mutant with pSdhA led to integration of the

vector at the SdhABE locus putting the complete SdhABE operon under the

control of the strong frd promoter. Further enzymatic activity measurements

proved that a real and active protein was produced.

In the same work, the compatibility of constructs with affinity tags for

detection was also tested. Juhnke et al. (2009) generated pSdhAHT and



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Michael Lafontaine and C. Roy D. Lancaster



pSdhAH1 by inserting oligonucleotide cassettes encoding a hexa-histidinetag with or without a following TEV-protease cleavage site at the start of the

sdhA gene or at amino acid position 37. It turned out that care must be taken

when using N-terminal affinity tags as only the AH1 variant with the hexahistidine-tag at position 37 could be detected in a Western blot with an antipenta-histidine antibody implicating that in the HT mutant, the N-terminal

his-tag was cleaved during export due the tat signal peptide corresponding to

amino acids 31–33 in SdhA. Furthermore, the use of a strepII-tag or a tandem strepII-tag for purification or detection of protein production was successfully demonstrated by Gross, Pisa, Saănger, Lancaster, & Simon (2004) or

Kern et al. (2010).

However, the frd promoter is not the only useable promoter for expression of foreign or modified genes. Gross and coworkers (Gross et al., 2004;

Gross, Simon, Theis, & Kroger, 1998) demonstrated the portability of the

procedure of generating deletion mutants and subsequent complementation.

They generated a deletion cassette harboring the kanamycin resistance gene

flanked by sequences homologous to the neighboring regions of the

HydABC operon. The following complementation with the pHydcat plasmid containing the HydABC operon with HydC variants as well as a chloramphenicol acetyltransferase gene yielded functional Fe/Ni hydrogenase.



2. METHODS

2.1 Materials

2.1.1 Ca/Mg solution (1000 ×)

Dissolve 0.74 g CaCl2 Á 2H2O and 5.1 g MgCl2 Á 6H2O in 100 mL

ddH2O. Autoclave and store at room temperature.

2.1.2 Cys/Glu solution (100×)

Dissolve 1 g glutamate and 0.69 g L-cysteine in 100 mL ddH2O. Autoclave

and store at room temperature.

2.1.3 Concentrate for nitrate media (20 ×) pH 7.5

Dissolve 121.0 g Tris, 80.8 g KNO3, 108.8 g sodium formate, 4.6 g

K2HPO4 Á 3H2O, 17.42 g K2SO4 and 11.6 g fumarate in 600 mL

ddH2O. Adjust to 1 L, autoclave, and add 4 mL trace elements solution after

cooling. Store at room temperature.



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