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Protein & Cell

, Volume 5, Issue 9, pp 658–672 | Cite as

The recombinant expression systems for structure determination of eukaryotic membrane proteins

  • Yuan He
  • Kan Wang
  • Nieng Yan
Open Access
Review

Abstract

Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryotic membrane protein structures have been obtained due to the technical challenges in the generation of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.

Keywords

eukaryotic membrane proteins recombinant expression structural biology integral membrane proteins (IMPs) fluorescence detected size exclusion chromatography (FSEC) protein purification and crystallization 

Abbreviations

β2AR

human β2 adrenergic G-protein-coupled receptor

ABCB10

ATP-binding cassette (ABC) transporters

AHA2

Arabidopsis thaliana auto-inhibited H1-ATPase 2

ASIC1

acid-sensing ion channel 1

CAAX protease Ste24p

C is cysteine redidue, A is an aliphatic residue and X is any residue. It is a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor

C8E4

tetraethyleneglycol monooctyl ether

C12E7

dodecylheptaglycol

C12E8

polyoxyethylene dodecyl ether

CHS

cholesteryl hemisuccinate

CmClC

cyanidioschyzon merolae chloride (Cl) ions transporter

C11Thio

n-undecyl-β-D-thiomaltopyranoside

CXCR4

human chemokine receptors

CX26

connexin 26 gap junction

CYMAL5

5-cyclohexyl-1-pentyl-β-D-maltoside

CYMAL6

6-cyclohexyl-1-hexyl-β-D-maltoside

CYMAL7

7-cyclohexyl-1-heptyl-β-D-maltoside

DDM

n-dodecyl-β-D-maltoside

DM

n-decyl-β-D-maltoside

FLAP

5-lipoxygenase-activating protein

GIRK2 (Kir3.2)

K+ channel: G protein-gated K+ channels

GluA2

a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive ionotropic glutamate receptor

GLuClα

caenorhabditis elegans glutamate-gated chloride channel a (GluCl), an inhibitory anion-selective Cys-loop receptor

HsAQP4

human aquaporin 4

HsAQP5

human aquaporin 5

Human BK channel

high-conductance voltage- and Ca21-activated K1 channels

K2P1

two-pore domain potassium (K+) channels

Kv1.2

voltage-dependent shaker family potassium channel

Kv1.2-Kv2.1 paddle

‘paddle-chimaera channel’, voltage-sensor paddle has been transferred from Kv2.1 to Kv1.2

LTC4S

cysteinyl leukotrienes

M-Ppase

membrane-integral pyrophosphatases

MAPEG

membrane-associated proteins in eicosanoid and glutathione metabolism

MNG

maltose-neopentyl glycol

NG

n-nonyl-β-D-glucopyranoside

NM

n-nonyl-β-D-maltoside

N/OFQ receptor

nociceptin/orphanin FQ receptor

OG

n-octyl-β-D-glucoside

OGNG

octyl glucose neopentyl glycol

P2X4

cation-selective ion channels gated by extracellular ATP

PAR1

protease-activated receptor 1

PfAQP

Plasmodium falciparum aquaglyceroporin

PiPT

a Fungal (Piriformospora indica) high-affinity phosphate transporter

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPE

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

POPG

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycero

RhCG

rhesus C glycoprotein

SoPIP2

1, spinach plant plasma membrane aquaporin

TRAAK

TWIK-related arachidonic acid–stimulated K+ channel

UDTM

n-undecyl-β-maltoside

UT-B

urea transporters-B

VrH+-Ppase

vigna radiate H1-translocating pyrophosphatases

ZMPSTE24

zinc metallopeptidase STE24

Introduction

It is estimated that approximately 30% of the protein-coding genes are for integral membrane proteins (IMPs) in human (Overington et al., 2006; Murray et al., 2012). IMPs are critical players for many important physiological processes including metabolism, signal transduction, and energy conversion and utilization (Krogh et al., 2001). Aberrant expressions and activities of IMPs are associated with a variety of diseases such as cancer, Alzheimer’s disease, and metabolic diseases (Ishikawa et al., 2004; Sanders and Myers, 2004; Overington et al., 2006; Aisenbrey et al., 2008; Bkaily and Al-Khoury, 2014). IMPs constitute more than 50% of the US Food and Drug Administration (FDA)-approved drug targets (Russell and Eggleston, 2000; Yildirim et al., 2007). Structures of eukaryotic membrane proteins are actively pursued for structure-based drug development.

In contrast to their physiological and pathophysiological significance, the progress on the structure biology of IMPs, particularly eukaryotic IMPs, has been relatively slow. By the end of March 2014, in total 466 unique membrane protein structures have been reported (Snider and Stephen, 2014), the majority of which are of prokaryotic origins. With respect to eukaryotic IMPs, more than half of the determined structures are for proteins obtained from endogenous sources (Bill et al., 2011). These proteins, exemplified by the electron transport chain complexes (Tsukihara et al., 1996; Xia et al., 1997; Sun et al., 2005), ATP synthases (Abrahams et al., 1994; Liu et al., 2004; Amunts et al., 2007), and photosystems (Kurisu et al., 2003; Liu et al., 2004; Amunts et al., 2007), usually exist in abundance and are biochemically stable, hence representing ideal candidates for structural analysis. However, the total types of endogenously abundant eukaryotic IMPs are limited. The majority of IMPs exist in low copies in the host species. Therefore, structural determination of most eukaryotic IMPs requires recombinant expression of the target proteins. The first atomic-resolution structure of a eukaryotic IMP obtained through recombinant expression, Kv1.2, was reported in 2005 (Long et al., 2005). Ever since, less than seventy structures have been obtained for eukaryotic IMPs generated through recombinant expression systems (Fig. 1).
Figure 1

The development trends in recombinant expression eukaryotic membrane proteins. The structure number of eukaryotic membrane protein is limited by some obstacles such as low yield and instability in detergents. Since the first eukaryotic membrane protein structure was determined in 2005, over sixty structures have been emerged until now

Out of the many challenges facing structural study of eukaryotic IMPs, production of sufficient quantities of well-behaved recombinant proteins represents the real technical bottleneck. Embedded in lipid bilayers, the structural integrity and proper functions of IMPs rely on the interactions with surrounding lipids (Phillips et al., 2009), which stabilize membrane proteins, provide lattice contacts, and in some occasions function as indispensable co-factors (van Meer et al., 2008). Recombinant expression of membrane proteins therefore requires a proper membrane environment. Whereas Escherichia coli proved to be the best host for most of prokaryotic IMPs of known structures, eukaryotic IMPs, with very few exceptions, requires eukaryotic expression systems including yeast, baculovirus-infected insect cells, and mammalian cells (Bill et al., 2011; Snider and Stephen, 2014).

In this review, in the hope of extracting some general principles on the expression and crystallization of eukaryotic membrane proteins, we examine the expression systems for the eukaryotic IMPs whose structures are obtained, attempt to summarize and compare the advantages and disadvantages of the representative recombinant expression systems, and delineate the detailed information in eukaryotic membrane protein purification and crystallization (Table 1).
Table 1

Expression, purification, and crystallization information for eukaryotic membrane proteins*

No.

Expression systems

Protein name

Family

Extraction detergent

Purification detergent

Gel filtration detergent

Final concentration (mg/mL)

Temperature (°C)

Methods

1

E. coli

Bl21(DE3)

FLAP

MAPEG

DDM

DDM

C12E8 + C8E4

  

Sitting drop

2

 

C43(DE3)

PfAQP

Water channel

OG

OG

OG

6

18

Hanging drop

3

 

Bl21(DE3)

Kir3.1-prokaryotic Kir channel chimera

Potassium channel

DDM

DDM

NG

8

20

Sitting drop

4

 

Bl21(DE3)

Cytochrome b561

Electron transport chain complexes

DM

NM

NG

 

18

Hanging drop

5

Yeast

Pichia Pastoris

Kv1.2 with β subunit

Potassium channel

DDM

DDM

DM

10

20

Hanging drop

6

  

Kv1.2-Kv2.1paddle

Potassium channel

DDM

DDM

CYMAL6 + CYMAL7

10

20

Hanging drop

7

  

Kv2.1paddle-Kv1.2 (F233 W)

Potassium channel

DDM

DDM

DDM

10

20

Hanging drop

8

  

Kir2.2 inward-rectifier

Potassium channel

DM

DM

DM

8

20

Hanging drop

9

  

GIRK2 (Kir3.2) K+ channel

Potassium channel

DM

DM

DM

6–7

20

Hanging drop

10

  

K2P1.1(KWIK-1)

Potassium channel

DDM

DDM

DDM

10

20

Hanging drop

11

  

K2P4.1(TRAAK)

Potassium channel

DDM

DDM

DM

5

4

Hanging drop

12

 

Pichia Pastoris

Calcium release-activated calcium channel

Calcium channel

DDM

DDM

NM + NG

16

17

Hanging drop

13

  

SoPIP2;1

Water channel

OG

OG

OG

10

4

Hanging drop

14

  

HsAQP5

Water channel

NG

NG

NG

10

8

Hanging drop

15

Yeast

 

HsAQP4

Water channel

OG

OG

OG

30

25

Hanging drop

16

  

P-Glycoprotein

ABC transporter

Triton

DDM

DDM

10

4

Sitting drop

17

  

P-Glycoprotein

ABC transporter

DDM

DDM

UDM

 

4

Hanging drop

18

  

LTC4S

MAPEG

DDM

DDM

DDM

6.5

4

Sitting drop

19

  

Histamine H1 receptor

GPCR

DDM

DDM

DDM

30–40

20

LCP

20

 

S. cerevisiae

AHA2 (H+ pump)

Pump

DDM

DDM

C12E8 + CYMAL5

20–30

4

Hanging drop

21

  

VrH+-Ppase

M-PPase

DDM

DM

DM

10

20

Hanging drop

22

  

NRT1.1

MFS transporter

DDM

DDM

DDM

10

4

Hanging drop

23

  

CAAX protease Ste24p

Intramembrane protease

DDM

DDM/C12E7

C12E7

7.35

4/17

Hanging drop

24

  

PiPT

MFS transporter

DDM

DDM

NG

10–15

20

Hanging drop

25

Insect cell

S. frugiperda

β2AR (Fab)

GPCR

DDM

DDM

DDM

8–12

22

Bicelle

26

  

β2AR (T4L)

GPCR

DDM

DDM

DDM

Concentrated

22

LCP

27

  

β2AR-agonist complex

GPCR

MNG

MNG

MNG (0.1%)

50

20

LCP

28

  

β2AR-GS complex

GPCR

MNG

MNG

MNG (0.1%)

90

20

LCP

29

  

A2A adenosine receptor

GPCR

DDM

DDM

DDM

70

20

LCP

30

  

CXCR4

GPCR

DDM

DDM

DDM

60–70

20

LCP

31

  

Dopamine D3 receptor

GPCR

DDM

DDM

DDM

20–30

20

LCP

32

  

Sphingosine 1-phosphate receptor

GPCR

DDM

DDM

DDM

100

20

LCP

33

  

M2 muscarinic acetylcholine receptor

GPCR

Digitonin + Na-cholate

DM

MNG

30

20

LCP

34

  

M3 muscarinic acetylcholine receptor

GPCR

DDM

DDM

MNG

60

20

LCP

35

  

κ-Opioid receptor

GPCR

DDM

DDM

DDM

40

20

LCP

36

  

μ-Opioid receptor

GPCR

DDM + CHAPS + CHS

DDM + CHAPS + CHS

MNG + CHS

30

20

LCP

37

  

δ-Opioid receptor

GPCR

MNG + CHAPS + CHS

MNG + CHAPS + CHS

MNG + CHAPS + CHS

50

20

LCP

38

Insect cell

S. frugiperda

N/OFQ receptor

GPCR

DDM + CHS

DDM + CHS

DDM + CHS

40

20

LCP

39

  

CCR5

GPCR

DDM + CHS

DDM + CHS

DDM + CHS

40–50

20

LCP

40

  

PAR1

GPCR

DDM + CHS

DDM + CHS

DDM + CHS

40–50

20

LCP

41

  

5-HT1B/2B serotonin receptor

GPCR

DDM + CHS

DDM + CHS

DDM + CHS

50–80

20

LCP

42

  

Smoothened receptor

GPCR

DDM + CHS

DDM + CHS

DDM + CHS

50–60

20

LCP

43

  

Glucagon receptor

GPCR

DDM + CHS

DDM + CHS

DDM + CHS

80

20

LCP

44

  

Metabotropic Glutamate Receptor 1

GPCR

DDM + CHS

DDM + CHS

DDM + CHS

50–80

20

LCP

45

  

P2X4

Channel

DDM

DDM

DDM

2

4

Hanging drop

46

  

ASIC1

Channel

DDM

DDM

DDM

5

4

Hanging drop

47

  

GluA2

Channel

DDM

DDM

C11Thio + lipids

2

4

Hanging drop

48

  

GLuCla

Cys-loop receptor

DDM

DDM

DDM

2

4

Hanging drop

49

  

CX26

Gap junction

DDM

DDM

UDM

30

4

Hanging drop

50

  

UT-B

Urea Transporter

DM

DM

OG

8

4

Sitting drop

51

  

ZMPSTE24

Intramembrane protease

DDM + CHS or OGNG + CHS

DDM + CHS or OGNG + CHS

DDM + CHS or OGNG + CHS

9–11

20

Sitting drop

52

Insect cell

 

ABCB10

ABC transporter

DDM

DDM

DDM

 

20

Sitting drop

53

  

Claudin-15

Tight junction

DDM

LMNG

LMNG

7

20

LCP

54

  

NRT1.1

MFS transporter

DDM

DDM

DDM

10

4

Hanging drop

55

 

Trichoplusiani

β1 adrenergic receptor

GPCR

DM

Octylthioglucoside

Octylthioglucoside

6

18

Hanging drop

56

  

NTS1 Neurotensin Receptor

GPCR

MNG + CHS

MNG + CHS

MNG + CHS

20–25

20

LCP

57

  

CmClC

H+/Cl- exchange transporter

DM

DM

DM

10

20

Hanging drop

58

  

Corticotropin-releasing factor receptor

GPCR

DM

DM

DM

20–30

22.5

LCP

59

  

GLUT1

MFS transporter

DDM

DDM

NG

10

4

Hanging drop

60

 

COS-1 cells

Rhodopsin

Rhodopsin

DDM

DDM

C8E4

   

61

Mammalian cell

HEK293

RhCG

Channel

OG

OG

OG

5

20

Hanging drop

62

 

Dopamine transporter

Solute carrier transpoter

DDM

DDM CHS POPC:POPE:POPG = 3:1:1

DM CHS POPE

3

4

Hanging drop

* The blank in the table is due to the details in the reported method has not been mentioned

Recombinant expression systems for eukaryotic membrane proteins

The recombinantly expressed eukaryotic IMPs of known structures were obtained from four systems: E. coli, yeasts (Pichia Pastoris and Saccharomyces cerevisiae), insect cells, and mammalian cells. These expression systems have their respective advantages and disadvantages. The choice of an appropriate expression system remains empirical, largely depending on the biochemical and biological properties of the target proteins (Bernaudat et al., 2011). Among the recombinantly expressed eukaryotic IMPs whose structures have been solved, 4 were expressed in E. coli, 20 in yeast, 35 in insect cells, and 3 in mammalian cells. Below we will discuss these four expression systems.

E. coli

As the most frequently exploited recombinant expression system, E. coli BL21 (DE3) has the obvious advantage of rapid replication, time-saving operation, inexpensive cost, and mature and easy genetic manipulations (Sahdev et al., 2008). E. coli C43 (DE3) and C41 (DE3) strains were developed for over-expression of membrane proteins (Miroux and Walker, 1996; Dumon-Seignovert et al., 2004). Indeed, these E. coli strains were employed to over-express the large majority of prokaryotic IMPs whose structures were finally obtained. However, as the prokaryotic expression systems, they may lack the essential lipids, molecular chaperons, and post-translational modifications that are required for the correct membrane insertion, folding, and function of eukaryotic IMPs (Sahdev et al., 2008). As a result, only 4 structures were obtained for eukaryotic IMPs expressed in E. coli (Table 2). Despite the challenge to express eukaryotic membrane proteins in E. coli, researchers attempted to overcome these hurdles with codon-optimization (Burgess-Brown et al., 2008) and protein fusion with Mistic or GlpF tag to promote protein expression (AegeanSoftware, 2005; Drew et al., 2006; Neophytou et al., 2007), and co-expression of post-translational machineries to facilitate protein folding (Mironova et al., 2005; Mijakovic et al., 2006). Regardless of the effort, E. coli may not be an ideal system for eukaryotic IMP expression.
Table 2

E. coli as an expression system for eukaryotic membrane protein

Expression systems

No.

Protein

Species

PDB code

Reference

E. coli

Bl21(DE3)

1

FLAP

Homo sapiens

2Q7 M 2Q7R

Ferguson et al., 2007

C43 (DE3)

2

PfAQP

Plasmodium falciparum

3C02

Newby et al., 2008

Bl21(DE3)

3

Kir3.1-prokaryotic Kir channel chimera

Streptomyces lividans

2QKS

Nishida et al., 2007

Bl21(DE3)

4

Cytochrome b561

Arabidopsis thaliana

4O6Y, 4O79, 4O7G

Lu et al., 2014

Yeast

Among the many yeast species, Pichia Pastoris (Pichia) and Saccharomyces cerevisiae (S. cerevisiae), which have been genetically well characterized, are the major systems to overexpress eukaryotic IMPs (Strausberg and Strausberg, 2001; Bornert et al., 2012). Schizosaccharomyces pombe is also employed for overexpression of IMPs, but not as widely as Pichia and S. cerevisiae (Yang et al., 2009). During the past thirty years, yeast has proved to be a useful expression system: 15 eukaryotic IMP structures have been determined for proteins expressed in Pichia expression system and 5 by S. cerevisiae. Most of the structurally available eukaryotic channels such as potassium channels and water channels were expressed in yeast, as listed in Table 3.
Table 3

Yeast as an expression system for eukaryotic membrane protein*

Expression systems

No.

Protein

Species

PDB code

Reference

Yeast

Pichia Pastoris

1

Kv1.2 with β subunit

Drosophila melanogaster

2A79

Long et al., 2005

2

Kv1.2-Kv2.1 paddle

Rattus norvegicus

2R9R

Long et al., 2007

3

Kv2.1paddle-Kv1.2 (F233 W)

Rattus norvegicus

3LNM

Tao et al., 2010

4

Kir2.2 Inward-Rectifier

Gallus gallus

3JYC

Tao et al., 2009

5

GIRK2 (Kir3.2) channel

Mus musculus

3SYO

Whorton and MacKinnon, 2011

6

K2P1.1 (KWIK-1)

Homo sapiens

3UKM

Miller and Long, 2012

7

K2P4.1 (TRAAK)

Homo sapiens

3UM7

Brohawn et al., 2012

8

Calcium release-activated calcium channel

Drosophila melanogaster

4HKR

Xiaowei Hou, 2012

9

SoPIP2;1

Spinacia oleracea

1Z98 2B5F

Tornroth-Horsefield et al., 2006

10

HsAQP5

Homo sapiens

3D9S

Horsefield et al., 2008

11

HsAQP4

Homo sapiens

3GD8

Ho et al., 2009

12

P-Glycoprotein

M. musculus

3G5U, 3G60, 3G61

Aller et al., 2009

13

P-Glycoprotein

Caenorhabditis elegans

4F4C

Jin et al., 2012

14

LTC4S

Homo sapiens

2PNO

Ago et al., 2007

15

Histamine H1 receptor

Homo sapiens

3RZE

Shimamura et al., 2011

S. cerevisiae

16

AHA2 (H+ pump)

Arabidopsis thaliana

3B8C

Pedersen et al., 2007

17

VrH+-Ppase

Vigna radiata

4A01

Lin et al., 2012

18

NRT1.1

Arabidopsis thaliana

4CL4

Parker and Newstead, 2014

19

CAAX protease Ste24p

Saccharomyces mikatae

4IL3

Pryor et al., 2013

20

PiPT

Piriformospora indica

4J05

Pedersen et al., 2013

* For some proteins like GPCR and potassium channel, only the representative ones are listed

Pichia is considered the best expression system among yeast species (Cereghino and Cregg, 2000). Several elements contribute to its increasing applications, including the simplicity of genetic manipulation, the high yield of heterologous protein, the cost-effective chemical reagents, as well as the ability of post-translational modifications (Macauley-Patrick et al., 2005). For these reasons, Pichia is a more suitable expression system for producing eukaryotic IMP than E. coli. Pichia shares the advantage of the molecular and genetic manipulation with S. cerevisiae, yet it adds extra advantage of 10- to 100- fold biomass out of the same cultural volume comparing with S. cerevisiae (Macauley-Patrick et al., 2005).

The improved techniques and the commercial availability together promote the development of Pichia (Cereghino and Cregg, 2000). Pichia is a methylotrophic yeast, capable of utilizing methanol as its sole carbon source (Yurimoto and Sakai, 2009). A promoter derived from the alcohol oxidase I (AOXI), which is the first-step enzyme in the methanol metabolism, strictly controls the expression of the foreign proteins (Macauley-Patrick et al., 2005). The commercial vector pPICZ (or pPICZα) takes advantage of the AOXI promoter, being induced by methanol (Li et al., 2007). AOXI promoter is prevailing than other promoters like PMA1 and GPD1 for its strong and highly inducible ability (Cereghino and Cregg, 2000). After the vector is readily prepared and transformed into the competent cells, the target gene can be inserted into the Pichia genome in high efficiency via homologous recombination to generate stable cell lines, and then the colonies with multiple copies that exhibit the highest protein expression level will be screened out through zeocin-spread plates (Daly and Hearn, 2005). This zeocin selective marker for transformation selection is important regarding to the convenience of genetic manipulation in yeast. All the procedure typically takes about 10–15 days for a complete procedure from subcloning to protein expression. A potential disadvantage of the yeast culture concerns the difficulty in cell disruption due to the thick and hard cell walls.

Insect cell

The baculovirus infected insect cell system is undoubtedly the dominant heterologous expression system for obtaining eukaryotic IMPs (Contreras-Gomez et al., 2014). The most common method for generating recombinant baculovirus is based on the site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) that is amplified in E. coli (Ciccarone et al., 1998). The process is very convenient: clone the target gene into the pFastBac vector which uses the strong AcMNPV polyhedron (PH) as the promoter for high level protein expression, then transform the pFastBac vector into DH10Bac E. coli competent cells. DH10Bac cells possess a baculovirus shuttle vector (bacmid) with a transposon site and a helper plasmid, thus can help the pFastBac vector to have a transposition on the bacmid. Once the transposition occurs and the recombinant bacmid is generated, the bacmid could be isolated and purified for transfection. After the insect cells are cultured into a desired confluence, they are transfected by the purified bacmid DNA to generate a recombinant baculovirus that used for preliminary expression test (Contreras-Gomez et al., 2014). The pFastBac is ampicillin resistance and Bacmid is kanamycin resistance, and these selective markers provide expedience for this baculovirus expression system. It takes approximately 3–4 weeks to complete these procedures for initial protein expression test.

There are two most popular insect cell lines used for IMP expression, Spodoptera frugiperda (Sf9) and Trichoplusia ni (Hi5). Heterologous proteins have disparate performances on the yield and behavior when expressed in these two cell lines (Unger and Peleg, 2012). Till now, 30 structures were obtained for eukaryotic IMPs from Sf9 expression system and 5 from Hi5 (Table 4).
Table 4

Insect cell as an expression system for eukaryotic membrane protein*

Expression systems

No.

Protein

Species

PDB code

Reference

Insect cell

S. frugiperda

1

β2AR (Fab)

Homo sapiens

2R4R 2R4S

Rasmussen et al., 2007

2

β2AR (T4L)

Homo sapiens

2RH1

Cherezov et al., 2007

3

β2AR-agonist complex

Homo sapiens

3PDS

Rosenbaum et al., 2011

4

β2AR-GS complex

Homo sapiens

3SN6

Rasmussen et al., 2011a,b

5

A2A adenosine receptor

Homo sapiens

3EML

Jaakola et al., 2008

6

CXCR4

Homo sapiens

3ODU 3OE8

Wu et al., 2010

7

Dopamine D3 receptor

Homo sapiens

3PBL

Chien et al., 2010

8

Sphingosine 1-phosphate receptor subtype 1

Homo sapiens

3V2 W 3V3Y

Hanson et al., 2012

9

M2 muscarinic acetylcholine receptor

Homo sapiens

3UON

Haga et al., 2012

10

M3 muscarinic acetylcholine receptor

Rattus norvegicus

4DAJ

Kruse et al., 2012

11

κ-Opioid receptor

Homo sapiens

4DJH

Wu et al., 2012

12

μ-Opioid receptor

Mus musculus

4DKL

Manglik et al., 2012

13

δ-Opioid receptor

Mus musculus

4EJ4

Granier et al., 2012

14

N/OFQ receptor

Homo sapiens

4EA3

Thompson et al., 2012

15

CCR5

Homo sapiens

4MBS

Tan et al., 2013

16

PAR1

Homo sapiens

3VW7

Zhang et al., 2012

17

5-HT1B/2B serotonin receptor

Homo sapiens

4IAR 4IB4

Wang et al., 2013a, b; Wacker et al., 2013

18

Smoothened receptor

Homo sapiens

4JKV

Wang et al., 2013a, b

19

Glucagon receptor

Homo sapiens

4L6R

Siu et al., 2013

20

Metabotropic glutamate receptor1

Homo sapiens

4OR2

Wu et al., 2014

21

P2X4

Danio rerio (Zebra fish)

3I5D 3H9 V 4DW1

Kawate et al., 2009; Hattori and Gouaux, 2012

22

ASIC1

Gallus gallus

2QTS 3HGC

Jasti et al., 2007; Gonzales et al., 2009

23

GluA2

Rat

3KG2 3KGC

Sobolevsky et al., 2009

24

GLuClα

Caenorhabditis elegans

3RHW, 3RIF, 3RI5 3RIA

Hibbs and Gouaux, 2011

25

CX26

Homo sapiens

2ZW3

Maeda et al., 2009

26

UT-B

Bos taurus

4EZC 4EZD

Levin et al., 2012

27

ZMPSTE24

Homo sapiens

4AW6

Quigley et al., 2013

28

ABCB10

Homo sapiens

4AYT

Shintre et al., 2013

29

Caludin-15

Mus Musculus

4P79

Suzuki et al., 2014

30

NRT1.1

Arabidopsis thaliana

4OH3

Sun et al., 2014

Trichoplusia ni

31

β1 adrenergic receptor

Meleagris gallopavo

2VT4

Warne et al., 2008

32

NTS1 Neurotensin Receptor

Rattus norvegicus

4GRV

White et al., 2012

33

CmClC

Cyanidioschyzonmerolae

3ORG

Feng et al., 2010

34

Corticotropin-releasing factor receptor

Homo sapiens

4K5Y

Hollenstein et al., 2013

35

GLUT1

Homo sapiens

4PYP

Deng et al., 2014

* For some proteins like GPCR and potassium channel, only the representative ones are listed

After the protein IL-2 was first expressed in large scale with the baculovirus-infected insect cells in 1985, this system has been quickly accepted and widely used (Smith et al., 1983; Maeda et al., 1985). Owing to the convenience of scale up, safety and accuracy (Kost et al., 2005), the baculoviral insect cell system has yielded the largest number of eukaryotic IMPs up to date (Table 4). Notably, among the 35 eukaryotic IMP structures, 23 are of G-protein coupled receptors (GPCR) (Table 4). The insect cell system has been the prevailing expression system for eukaryotic IMP. However, the cost for the cultural medium may represent a serious roadblock for most laboratories.

Mammalian cell

Mammalian expression system has become one of the popular recombinant protein production systems for its proper post-translational modification and human protein-like structure assembly (Khan, 2013). HEK (human embryo kidney) and CHO (Chinese hamster ovary) are two broadly used cell lines for recombinant expression. These two cell lines are extensively applied by researchers to do functional assay such as the electrophysiological assay (Kawate et al., 2009). Both these two cell lines can be applied for transient and stable transfections (Zhu, 2012). For the transient transfection approach, it is relative easier to reach to a reasonable protein expression level, but this expression level may vary from batch to batch. On the other hand, although the proteins have higher productivity and less variation in the stable transfection method, it is very time consuming (one month at least) (Condreay et al., 1999; Baldwin et al., 2003). Consequently, it is a balance for scientists to choose between these two transfection methods.

HEK293 is a specific cell line originally derived from HEK cells, while the number “293” comes from Graham’s habit of numbering his experiments (Louis et al., 1997). Large scale, transient transfection of HEK293 in suspension culture is a reliable way to generate milligram quantities of recombinant eukaryotic IMPs. When the gene of interest is ligated into the vector pcDNA3 or pCMV5, the complete plasmid is then transfected into the HEK293 cells and the cells are harvested after 48 h (Thomas and Smart, 2005). The whole procedure is more or less similar to that of the insect cell system, only with a couple of exceptions. For example, 5%–10% CO2 is required for maintaining the HEK293 cells, and the culture temperature is 37°C for HEK293 but not 27°C as for insect cells. The overall process usually requires one to two weeks from initial cloning to small scale test for the transient expression. However, ascribe to the low yield, slow growth rate and higher cost of complex media (Sunley and Butler, 2010), the number of eukaryotic IMP structures generated based on the mammalian cells is very limited. So far, only three eukaryotic IMP structures are from this system, and two of them are obtained from HEK293 cells (Table 5).
Table 5

Mammalian cell as an expression system for eukaryotic membrane protein

Expression system

No.

Protein

Species

PDB code

Reference

Mammalian

HEK293

1

Rodopsin

Homo sapiens

2J4Y

Standfuss et al., 2007

2

RhCG

Homo sapiens

3HD6

Gruswitz et al., 2010

3

Dopamine transporter

Homo sapiens

4M48

Penmatsa et al., 2013

The BacMam system has to be mentioned for its safety, reproducibility and efficiency (Dukkipati et al., 2008). The baculoviruses are engineered by inserting a mammalian expression cassette for delivering foreign genes in mammalian cells. Their non-replicating property makes they are safe and well-tolerated by mammalian cells. BacMam system gains widespread use for its safety and rapid manipulation (Reeves et al., 2002; Baconguis and Gouaux, 2012). Depending on the cell type, cell division rate and transduction efficiency, it lasts 5–14 days to detect the gene expression (Dukkipati et al., 2008). The dopamine transporter structure was determined by the BacMam system (Penmatsa et al., 2013).

From the foregoing discussion, it is concluded that every expression system has their distinctive properties for protein expression. We compare their relative merits for an intuitional understanding of each system which can help researchers to make the best choice for their proteins expression (Table 6).
Table 6

Comparison among four expression system

 

E. coli

Yeast (Pichia)

Insect cell (Sf9)

Mammalian cell (HEK293)

Duration time before cell cultivation (Days)

3–5

6–8

25–30

Transient: 3–5 Stable: at least 30

Cell cultivation time for 1L test (Days)

1–2

3–7

2–4

2–4

Cost for 1L test ($) in China

15–20

20–25

200–250

200–250

Number of available eukaryotic IMP structures

4

20

34

3

Homologue screen

Eukaryotic membrane proteins are very difficult to yield in large quantities, and most of them tend to be unstable in the presence of detergents. As a result, identification of well-expressed proteins is very essential. Homologue screen is widely applied for researchers to discover well-behaved proteins (Kawate et al., 2009; Xiaowei Hou, 2012).

Fluorescence detected size exclusion chromatography (FSEC) is a powerful method for homologue screen (Drew et al., 2006; Newstead et al., 2007). Compared with the common protocols, GFP fusion membrane proteins can be detected by measuring fluorescence in whole cells during the over-expression process. It saves time to help people preclude proteins that have no expression or low expression level. Also, it is much easier to assess the integrity of proteins by detecting the fluorescence in SDS polyacrylamide gels. Moreover, FSEC could be employed to figure out the most stable detergents in initial detergent screen. Considering these benefits, this technology is very widely applied (Jasti et al., 2007; Gonzales et al., 2009; Kawate et al., 2009; Sobolevsky et al., 2009). Taking P2X receptor as an example (Kawate et al., 2009), because of its aggregation and instability problems, researchers applied this method to screen 35 orthologs and finally got one species which was fit for crystallization. FSEC is proven to be one of the most robust methods to facilitate the identification of appropriate candidates for solving the structures of eukaryotic membrane proteins.

Optimal constructs design

Optimizing constructs is very beneficial for getting the well-packed crystals. One way for optimizing constructs is to “cut off”. Limited proteolysis is a conventional method to find the optimal constructs. Besides, it is worth noting that either N-terminal tag or C-terminal tag is removed before crystallization in most crystallization cases (Long et al., 2005; Long et al., 2007; Gonzales et al., 2009; Maeda et al., 2009; Sobolevsky et al., 2009; Tao et al., 2009). For instance, the desensitized ASIC1 was crystallized by removal of 25 N-terminal and 64 C-terminal residues (Jasti et al., 2007).

The contrary way for optimizing constructs is to “add up”. T4 lysozyme (T4L) insertion and Fab/nanobody replacement are applied to produce stable proteins. The T4L fragment is soluble enough to effectively increase the solvent-exposed area, thereby facilitating protein-protein interactions and generating novel crystal packing interfaces (Cherezov et al., 2007). Fab/nanobody, which are generated from monoclonal antibodies, can reduce the protein flexibility and improve the conformational homogeneity (Zhou et al., 2001; Rasmussen et al., 2007). GPCR is one of the most successful cases employing T4L and Fab/nanobody to the ultimate structure determination (Rasmussen et al., 2007; Rasmussen et al., 2011a, b).

Mutagenesis is an alternative way for constructs design. In order to improve the crystallization behavior and stabilize the tetrameric state of the glutamate receptor GluA2, point mutations were introduced, preventing non-specific aggregation and disulphide bond formation (Sobolevsky et al., 2009). And E329Q was introduced in order to stabilize GLUT1 in a certain conformation (Deng et al., 2014). Plus, glycosylation is the most common post-translational modification of eukaryotic membrane proteins and leads to heterogeneity of proteins. Thus, mutating of glycosylation sites or deglycosylation by enzymes is an essential step for crystallization (Deng et al., 2014).

Detergents, lipids and crystallization

We have summarized the detergents used for protein purification and crystallization from Table 1. 51 eukaryotic membrane proteins can be extracted from DDM or DM (Fig. 2A), suggesting DDM/DM are the detergents suitable for the extraction process of the majority of eukaryotic membrane proteins. Collaterally, nearly half of the eukaryotic membrane protein crystals are obtained from DDM/DM, indicating DDM/DM are worthy of a trial for crystallization in the first place (Fig. 2B and Table 1). Apart from these conventionally applied detergents, new detergents have also been developed to meet the new requirements. For example, when purifying β2 adrenergic receptor-Gs protein, the authors stabilized protein complex by exchanging DDM with a newly developed maltose neopentyl glycol detergent MNG-3 (NG310, Anatrace) to prevent the complex dissociated from original detergent DDM (Chae et al., 2010; Rasmussen et al., 2011a, b).
Figure 2

Detergents used for extraction and crystallization of eukaryotic membrane proteins. (A) Detergents for protein extraction and purification. DDM/DM can be applied for most eukaryotic membrane proteins in extraction step. (B) Detergents for protein crystallization. DDM/DM is the major detergent for the crystallization of eukaryotic membrane proteins

It is worth noting that additional lipids are able to help crystal packing. There are three ways of lipid combinations. The first is mixing lipids with detergent(s) in hanging or sitting drop during crystallization. Take mammalian voltage-dependent shaker family potassium channel as an example, the author utilized 0.1 mg/mL 3:1:1 POPC: POPE: POPG throughout purification and crystallization to obtain crystals (Long et al., 2005). The second approach is lipid cubic phase (LCP) method. The lipid cubic phase is a dynamic structure consisting of a highly organized single lipid bilayer pervaded by an inter-connected aqueous channel (Landau and Rosenbusch, 1996). Martin has an elaborate discussion about LCP method which we will not go into details in this review (Caffrey and Cherezov, 2009). The crystal structure of β2AR-GS complex was determined by the use of 7.7 MAG as the host lipid for crystallization (Rasmussen et al., 2011a, b). The third way is bicelle method, which is regarded as an intermediate approach between the traditional detergent crystallization method and the rigid LCP method. Bicelle can be considered as a lipid bilayer disc that formed by a long chain lipid and a short chain lipid or detergent (Agah and Faham, 2012). The general composition is 3:1 DMPC: CHAPSO. Several protein structures were determined utilizing bicelle method (Rasmussen et al., 2007; Payandeh et al., 2011).

Last but not the least, we will elaborate a few messages for the crystallization of eukaryotic membrane protein drawn from Table 1: (a) Protein concentration: almost all the protein concentration for crystallization is above 5 mg/mL. (b) Crystallization temperature: if we expel the LCP method that is routinely crystallized at 20 ± 2°C, nearly half of the eukaryotic membrane proteins are crystallized at low temperature, especially on 4°C. At cold temperature, for protein with “normal” solubility, protein will be more soluble in high salt and precipitate from lower concentration of the precipitant reagents, and also the equilibrium diffusion rate occurs more slowly. These manifest that crystallization at lower temperature is absolutely an indispensable trial. (c) Crystallization methods: hanging drop or sitting drop crystallization method is the main and conventional approach for most eukaryotic membrane protein. LCP method is an up-rising star which is extensively applied in determining the GPCR’s structures which we have mentioned before. Remarkably, LCP method is not only propitious to GPCR, but also is able to be applied for none-GPCR protein structures determination (Suzuki et al., 2014).

Conclusion

In this review, we discuss the benefits and drawbacks of different expression systems for eukaryotic membrane protein, and illustrate some general methods of recent advances for eukaryotic membrane protein purification and crystallization. We hope our work can provide help to those who are interested and work on eukaryotic membrane proteins. Although the discussion of eukaryotic membrane protein structure determined by Cryo-EM or NMR is beyond the scope of this review, the general methodologies and technical strategies summarized here also come to an aid in protein yield augment and sample homogeneity improvement for Cryo-EM and NMR. They are very powerful tools to solve structures, for instance, the Cryo-EM was applied to determine TrpV1 structures (Cao et al., 2013; Liao et al., 2013). With the development of advanced technologies, more and more eukaryotic membrane protein structures will emerge to answer the most significant questions in life sciences and provide the novel pharmaceutical targets in drug design.

Notes

Acknowledgements

We apologize to colleagues whose work could not be cited due to the scope of this review. We would like to thank members in Yan laboratory for discussions. We thank Brendan Lehnert, Xinlei Sheng, Quanxiu Li, Dan Ma and Xinhui Zhou for critical reading. This work was supported by funds from the National Basic Research Program (973 Program) (No. 2011CB910501), the National Natural Science Foundation of China (Grant Nos. 31321062-20131319400, 31125009, and 91017011), and funds from Tsinghua-Peking Center for Life Sciences. The research of N.Y. was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute.

Compliance with Ethics Guidelines

Yuan He, Kan Wang, and Nieng Yan declare that we have no conflict of interest.

This review does not contain any studies with human or animal subjects performed by the any of the authors.

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Authors and Affiliations

  1. 1.State Key Laboratory of Bio-membrane and Membrane BiotechnologyTsinghua universityBeijingChina
  2. 2.Center for Structural BiologySchool of Medicine, Tsinghua universityBeijingChina
  3. 3.China-Japan Friendship HospitalBeijingChina

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