Heterologous Membrane Protein
Expression and Purification from
Pyrococcus furiosus
Han-Seung Lee, Michi Izumi, Francis E. Jenney Jr, Farris Poole,
Frank Sugar, Claudia Shah, Michael W.W. Adams
Southeast Collaboratory for Structural Genomics (SECSG),
University of Georgia,
Pyrococcus furiosus
• Optimal growth at 100°C (range 70-105°C)
• Genome is 1.908 Mb and fully sequenced
• 2,197 putative single open reading frames (sORFs)
• All ORFs were cloned N-terminal His-Tag vector
Transmembrane Domain(TMD) prediction
programs: PRED-TMR2, TSEG, SOSUI
Signal sequence prediction programs:
SignalP, SOSUI, TargetP
TMDs by PRED-TMR2
1,782 - no TMDs (80%)
452 - TMDs
(20%)
139 - 1 TMD
(6%)
313 - > 1 TMD (14%)
Signal Sequences by SignalP
389 - SS
(18%)
TMDs + Signal Sequences
271 - both
(12%)
Transporter related ORFs in P. furiosus predicted by TransportDB
Type (total ORFs)
ATP-dependent
(116 ORFs)
Ion channel
(5 ORFs)
Secondary transporter
(70 ORFs)
Unclassified
(4 ORFs)
Superfamily/Family
ATP Binding Cassette (ABC)
F-ATPase, V-ATPase, A-ATPase
P-ATPase
Metal ion transporter (MIT)
Small conductive mechanosensitive ion channel (MscS)
Voltage-gated ion channel (VIC)
Arsenical Resistance-3 (ACR3)
Auxin Efflux Carrier (AEC)
Alanine or Glycine:Cation symporter (AGCS)
Arsenite-Antimonite (ArsB) Efflux
Ca2+:Cation Antiporter (CACA)
Cation diffusion facilitator (CDF)
Monovalent cation:Proteon Antiporter-2 (CPA2)
Monovalent cation (K+ or Na+):Proteon Antiporter-3 (CPA3)
Dicaboxylate/Amino acid:cation(K+ or Na+) symporter (DAACS)
Divalent Anion:Na+ symporter (DASS)
Drug/Metabolite transporter (DMT)
Major Facilitator Superfamily (MFS)
Multidrug/Oligosaccharidyl-lipid/Polysaccharide (MOP) Flippase
Nucleobase:cation symporter-1 (NCS1)
Nucleobase:Cation Symporter-2 (NCS2)
Na+:H+ Antiporter (NhaC)
Neurotransmitter:Sodium symporter (NSS)
Inorganic phosphater transporter (PiT)
Resistance-nodulation-cell division (RND) superfamily
Solute:sodium symporter (SSS)
K+ transporter (TrK)
Ferrous Iron uptake (FeoB)
Mercuric ion permease (MerTP)
Magnesium ion transporter-E (MgtE)
Total transporter related ORFs : 195 ORFs (103 transporters)
ORFs
105
9
2
1
2
2
1
1
1
1
1
2
4
21
1
1
7
10
5
1
2
1
2
2
3
1
2
2
1
1
Fifty ORFs that did not expressed in E. coli BL21 (DE3) were introduced
into C41(DE3) and C43(DE3)
SSE test of membrane associated protein (Project 28)
- All selected 50 genes were not expressed in E. coli BL21 (DE3) in SSE or LSE
*
* TMD avg = average of TMDs predicted by three different TMD prediction program (TSEG, SOSUI,PRED-TMR2)
** Expression was detected by ELISA
*
Mid scale expression test of membrane proteins
50 ORFs were expressed in C41 and C43
ELISA
SSE (Small scale expression, 1 ml culture)
50.0%
25 proteins showed positive signals in C41 and C43 strains
(4 different conditions)
12 ORFs were selected and expressed in 30 ml culture
SDS-PAGE
83.3%
10 proteins were expressed in 30 ml culture
6 ORFs were selected and fractionated from membrane
SDS-PAGE
2 proteins were expressed in membrane fraction
(Overall success rate ≈ 14%)
33.3%
SSE * of membrane proteins (Project 29 & 30)
178 proteins that have 3 TMDs at least were selected and expressed in 30ml scale
Project 29
1
2
3
4
5
6
7
8
9
10
11
12
A
PF1263
PF0824
PF1856
PF1157
PF0790
PF1240
PF0170
PF0740
PF2049
PF1937
PF1617
PF0698
B
PF0429
PF1520
PF0503
PF1164
PF0910
PF1469
PF0036
PF1677
PF0004
PF1968
PF1654
PF1876
C
PF1446
PF0897
PF0730
PF0552
PF1371
PF0857
PF1106
PF0148
PF0278
PF0419
PF2013
D
PF0143
PF0371
PF0411
PF0783
PF0046
PF0834
PF1409
PF0745
PF0350
PF1252
PF0045
E
PF0826
PF0443
PF0109
PF1292
PF1697
PF0317
PF1726
PF0578
PF0947
PF0268
PF0234
F
PF0852
PF0981
PF1585
PF1350
PF0663
PF0486
PF1963
PF0582
PF1671
PF0392
PF0993
G
PF1336
PF1092
PF0275
PF1354
PF0106
PF1006
PF0191
PF0883
PF0394
PF0705
PF0174
H
PF0514
PF1447
PF1020
PF1603
PF0520
PF2043
PF0044
PF1888
PF1247
PF1936
PF0519
Project 30
1
2
3
4
5
6
7
8
9
10
11
12
A
PF1984
PF1144
PF1211
PF1749
PF1727
PF1845
PF0192
PF0326
PF1844
PF1775
PF0529
PF1000
B
PF1228
PF0746
PF1740
PF0281
PF0130
PF1303
PF0744
PF0816
PF1064
PF0398
PF0777
PF1058
C
PF0531
PF0831
PF1001
PF0665
PF0237
PF0081
PF1372
PF1256
PF1081
PF0659
PF0091
D
PF0915
PF0922
PF0167
PF0949
PF0457
PF2037
PF0712
PF1452
PF1235
PF0040
PF0416
E
PF0163
PF1465
PF0309
PF0129
PF0230
PF1373
PF1425
PF0648
PF1595
PF0134
PF1923
F
PF0718
PF1524
PF1428
PF0623
PF1514
PF1135
PF1426
PF0697
PF1841
PF0977
PF0365
G
PF1887
PF0117
PF0515
PF1089
PF0299
PF1325
PF1453
PF1448
PF0684
PF1575
PF0465
H
PF1007
PF0992
PF0558
PF1494
PF1075
PF1073
PF1799
PF1737
PF0896
PF1846
PF0729
* Expression was detected by ELISA
178 membrane proteins  32 proteins were expressed (17%)
Purification procedure for membrane protein
Fermentation (5-20L)
Sonication
Membrane fractionation
Heat treatment
His-tag purification
Ion-ex chromatography
Gel filtration
(Using Triton X-100)
(Optional)
(Twice – detergent change)
(Optional)
Membrane fractionation
Sonication (0.2g wet cell/ml)
Centrifuge
Supernatant
Inclusion body & Undisrupted
Ultracentrifuge
Supernatant
Ppt. (Membrane & IB)
Wash membrane pellet 2 x with buffer
Ultracentrifuge after each wash
Dissolve membrane with buffer containing 1 % Triton-X
Stir at 4°C for 3h at least
Ultracentrifuge
Supernatant
Membrane fraction
Ppt.
His-tag purification of PF0816
1st purification (Triton X-100)
Western blot
kDa
177.3
110.7
79.8
61.0
47.8
35.9
24.5
18.7
13.9
SDS-PAGE
2nd Purification (OG)
SDS-PAGE
Gel Filtration (Superdex-75) of PF0816
(under 0.8% octyl-β-D-glucopyranoside)
SDS-PAGE
2003Aug05no001:1_UV
2003Aug05no001:1_Fractions
SDS-PAGE
2003Aug05no001:1_Logbook
mAu
Fraction # 16
70.0
kDa
177.3
60.0
110.7
50.0
79.8
61.0
40.0
47.8
35.9
30.0
concentrated
24.5
18.7
20.0
13.9
10.0
5.9
Method Run 8/5/2003, 10:21:07 AM, Method : , Result : D:\...\prime\2003Au
Break point 4
0.0
123456789 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79
0
20
40
60
80
100
120
Waste
140
min
Total amount = 1.33mg
(From 5 L)
Quality control using MALDI analysis (PF0816)
MVALDEPLPY VGVTPMQVLT AIVVLIVGYI VAKVVVASFK
RGLKKTKLPE LVVEFLGRFL SALLYVAVIL LAVRALGIEV
GSVVLGLSAV IGLILGFGMQ DTLTNLAAGV WIAALRPIDI
GEVVEVAGKV GKVNAVGIMS TELLTADNVL ITIPNKLVWG
NVITNYTRMP TRRVDVNVGV AYGTDLDKAI KVAMELMQNH
PKVLKDPAPA VVVTELGDSS INLQLRAWAK TEDYWTVKFD
LTKGIYEAYR REGIEIPFPQ LDVHIKEMPK
Underlined sequence : Predicted TMDs
Red sequence : Peptides from MALDI
Mw
871.2820
1435.5930
1734.6980
2193.9040
2534.1320
Mw in DB Difference
871.4314 -0.1494
1435.7698 -0.1768
1734.9430 -0.2450
2194.1719 -0.2679
2534.4194 -0.2874
A. acid sequence
244 250 (K) GIYEAYR(R)
157 168 (K) LVWGNVITNYTR(M)
252 266 (R) EGIEIPFPQLDVHIK(E)
206 226 (K) DPAPAVVVTELGDSSINLQLR(A)
203 226 (K) VLKDPAPAVVVTELGDSSINLQLR(A)
0 unmatched masses: The matched peptides cover 21% (58/270 AA's) of the protein.
Coverage Map for This Hit (MS-Digest index #): 887
Crystals of PF0816
The protein crystallized in 12 % PEG 4000
100 mM NaCl
100 mM Lithium Sulfate
100 mM HEPES (pH 7.6)
Conclusions
1. C41(DE3) and C43(DE3) are useful host for membrane protein
expression and C43(DE3) is better than C41(DE3).
2. Membrane proteins were purified using the “crystallography-favored”
detergent, octyl-β-D-glucopyranoside (OG).
3. If membrane proteins were sufficiently expressed in membrane, three
can be purified within a week.
Work in progress
1. Membrane proteins with signal peptide
 expression in C-terminal His-tag vector
2. Optimization of expression condition
 Autoinduction media and optimization of fermentation condition
3. Membrane protein complex
 Coexpression of membrane protein complex