Structural genomics of human proteins – target selection and generation of a public catalogue of expression clones

1475-2859-4-21 1475-2859 Research Structural genomics of human proteins – target selection and generation of a public catalogue of expression clones Büssow Konrad buessow@molgen.mpg.de Scheich Christoph scheich@molgen.mpg.de Sievert Volker volker@volkerspage.de Harttig Ulrich ulrich.harttig@arcor.de Schultz Jörg Joerg.Schultz@biozentrum.uni-wuerzburg.de Simon Bernd simon@embl-heidelberg.de Bork Peer Peer.Bork@EMBL-Heidelberg.de Lehrach Hans lehrach@molgen.mpg.de Heinemann Udo heinemann@mdc-berlin.de

Protein Structure Factory, Heubnerweg 6, 14059 Berlin, Germany

Max-Planck-Institut für Molekulare Genetik, Ihnestr. 73, 14195 Berlin, Germany

EMBL Heidelberg, Meyerhofstr. 1, 69117 Heidelberg, Germany

RZPD German Resource Center for Genome Research GmbH, Heubnerweg 6, 14059 Berlin, Germany

DIFE, Arthur-Scheunert-Allee 114–116, 14558 Nuthetal, Germany

Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, 13092 Berlin, Germany

Institut für Chemie/Kristallographie, Freie Universität, Takustr. 6, 14195 Berlin, Germany

Department of Bioinformatics, University of Würzburg, Biocenter, Am Hubland, 97074 Würzburg, Germany

Microbial Cell Factories 1475-2859 2005 4 1 21 http://www.microbialcellfactories.com/content/4/1/21 15998469 10.1186/1475-2859-4-21 23 5 2005 05 7 2005 05 7 2005 2005 Büssow et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The availability of suitable recombinant protein is still a major bottleneck in protein structure analysis. The Protein Structure Factory, part of the international structural genomics initiative, targets human proteins for structure determination. It has implemented high throughput procedures for all steps from cloning to structure calculation. This article describes the selection of human target proteins for structure analysis, our high throughput cloning strategy, and the expression of human proteins in Escherichia coli host cells.

Results and Conclusion

Protein expression and sequence data of 1414 E. coli expression clones representing 537 different proteins are presented. 139 human proteins (18%) could be expressed and purified in soluble form and with the expected size. All E. coli expression clones are publicly available to facilitate further functional characterisation of this set of human proteins.

Background

The Protein Structure Factory

The Protein Structure Factory (PSF) is a joint endeavour of universities, research institutes and companies from the Berlin area 12. It takes part in the international structural genomics initiative 34 and aims at the determination of human protein structures by X-ray diffraction methods and NMR spectroscopy using standardised high-throughput procedures. A complete pipeline has been established for this purpose that comprises cloning, protein expression in small and large scale, biophysical protein characterisation, crystallisation, X-ray diffraction and structure calculation.

It is known that eukaryotic proteins are often difficult to express in Escherichia coli 5. Only a certain fraction of these proteins can be overproduced in E. coli in sufficient yield without formation of inclusion body aggregates or proteolytic degradation. Alternative expression systems include cell cultures of various eukaryotic organisms and cell-free, in vitro protein expression. These systems have been greatly improved since 1999, when the PSF project was initiated. In the meantime, E. coli 567 and wheat germ 8in vitro protein synthesis is routinely used by structural genomics projects. At the PSF, yeast expression hosts, Saccharomyces cerevisiae and Pichia pastoris, were successfully established as alternative systems to E. coli, as described in detail previously 91011. We will focus here on the results obtained with the E. coli expression system.

E. coli strains and vectors

The T7 RNA polymerase-dependent E. coli expression vector system (pET-vectors) is a universal system to generate recombinant protein for structural analysis 12. pET vectors are usually combined with the E. coli B strain BL21 and derivatives that are engineered to carry the T7 RNA polymerase gene. These strains, however, have limitations in cloning and stable propagation of the expression constructs. Expression vectors which are regulated by the lac operator are independent of the host strain. Recombination-deficient E. coli K-12 strains are suitable for cloning because of their high transformation rates and because they allow for stable propagation of recombinant constructs. The strain SCS1 (Stratagene; hsdR17(r_K^-m_K⁺) recA1 endA1 gyrA96 thi-1 relA1 supE44) was found to perform well at the PSF in cloning experiments. It grows relatively fast and allows for robust protein expression.

Affinity tags allow for standardised protein purification procedures. The first vector that was used routinely in the PSF, pQStrep2 (GenBank AY028642, Figure 1), is based on pQE-30 (Qiagen) and adds an N-terminal His-tag 13 for metal chelate affinity chromatography (IMAC) and a C-terminal Strep-tag II 1415 to the expression product. pQStrep2 allows for an efficient two-step affinity purification of the encoded protein, as demonstrated in a study of an SH3 domain 16. The eluate of the initial IMAC is directly loaded onto a Streptactin column. Thereby, only full-length expression products are purified and degradation products are removed. However, the two tags, which are flexible unfolded peptides, remain on the protein and may interfere with protein crystallisation, although we could show that crystal growth may be possible in their presence even for small proteins 16. To exclude any negative influence by the affinity tags, another vector, pQTEV (GenBank AY243506, Figure 1), was constructed. pQTEV allows for expression of N-terminal His-tag fusion proteins that contain a recognition site of the tobacco etch virus (TEV) protease for proteolytic removal of the tag.

Figure 1

Vector maps

Vector maps. Vector maps of pQStrep2, pQTEV and pSE111

Codon usage has a major influence on protein expression levels in E. coli 17, and eukaryotic sequences often contain codons that are rare in E. coli. Especially the arginine codons AGA and AGG lead to low protein yield 18. This can be alleviated by introducing genes for overexpression of the corresponding tRNAs into the E. coli host cells. We have used the plasmid pSE111 (Figure 1) carrying the argU gene for this purpose. pSE111 is compatible with pQTEV and other common expression vectors. It carries the lacI^Qgene for overexpression of the Lac repressor, which is required when using promoters regulated by lac operators. pSE111 was used at the PSF in combination with the expression vectors pQStrep2 and pGEX-6P-1. Strains for overexpression of rare tRNAs are available from Invitrogen (BL21 Codon Plus) and Novagen (Rosetta). The Rosetta strain contains the chloramphenicol-resistant pRARE plasmid that supplies tRNAs for the codons AUA, AGG, AGA, CUA, CCC, GGA 19. This plasmid is used at the PSF in combination with pQTEV and pGEX-6P-2.

The Additional file 1 psfClones.xml lists the vector and helper plasmid for overexpression of rare tRNAs that was used for each individual clone.

Selection of target proteins

We selected target proteins with higher-than-average chances of successful expression in E. coli and crystallisation 1. Proteins were excluded for which sequence analysis predicted that structure determination would be difficult. Starting from the complete set of known human proteins, potentially difficult target proteins and proteins of known structure were excluded according to the following criteria:

• Membrane proteins are known to be complicated targets for structure determination and were excluded. Membrane proteins were identified with the program TMHMM 2021.

• Since very large proteins are often difficult to express, the maximal length of target proteins was set to 500 amino acids.

• Protein regions that are unstructured or only partially structured 22 may lead to difficulties during protein expression and purification. Unstructured regions are susceptible to proteolyic attack, and represent an obstacle to protein crystallisation. A large proportion of intrinsically unstructured protein sequences are characterised by sequence stretches of low complexity and tandem repeats 23. Proteins with low complexity regions of more than 20 amino acids length, detected by the SEG program, or with more than one region were excluded 2425.

• Coiled-coil proteins were excluded from our target list, since this fold is not novel, and structural analysis of coiled coils requires special attention. Many coiled coil proteins form hetero-complexes with other coiled-coil proteins and cannot be studied without their binding partner. Coiled-coils domains are long, extended structures which can usually only be crystallised as domains, i.e. expression constructs lacking other domains have to be prepared. To identify coiled-coil proteins, the program COILS was used 262728.

• The cellular localisation of target proteins was assigned with the Meta_A(nnotator) 2930. Target proteins annotated to be localised in the extracellular space, endoplasmic reticulum, Golgi stack, peroxisome or mitochondria were excluded from expression in E. coli. Many of these proteins require formation of disulphide bonds for correct folding, but these are generally not formed in the reducing environment of the E. coli cytoplasm. Therefore, these proteins were allocated only for extracellular expression by yeast host cells. Proteins with predicted intracellular localisation or which were not assigned with a localisation by Meta_A(nnotator) were expressed in the cytosol of E. coli.

• Potential target proteins were matched to the sequences of proteins with known structure at the Protein Data Bank (PDB) 31. PSI-BLAST 32 was used to detect even very distinct homologies to PDB entries to rule out proteins with known folds. This filter was later replaced by a less stringent one, which considers the sequence identity and 'coverage' of matches to PDB sequences. The coverage is the length of the sequence match divided by the protein length. According to the less stringent filter, proteins with 60% or more sequence identity over 90% or more of the sequence length were excluded. Thereby target proteins could be included of which only a part, e.g. a single domain, has a known structure.

Results

Target lists

The first target list was generated in 1999, when the PSF project started, from the set of all human proteins known at that time. This set was filtered as described above. PSI-BLAST was used to match potential target proteins to the PDB and to include only proteins of presumably novel folds. To enable high-throughput cloning, we selected target proteins for which full-length cDNA clones were available. In 1999, cDNA clones of the IMAGE consortium represented the main public source of sequenced cDNA clones 33. However, only partial sequence information existed for these clones – the EST sequences of the dbEST database 34 – and only a small proportion contained complete open reading frames (full-ORF clones). 490 proteins were selected which met the selection criteria, had no match to PDB detected by PSI-BLAST and for which full-ORF IMAGE cDNA clones were available.

In 2003, a second target list was compiled from novel full-length cDNAs discovered by the German cDNA consortium 3536. The same filter criteria as for the first target list were applied, except that cellular localisation was not taken into account. Proteins with a PDB match of 60% or more sequence identity and 90% sequence coverage were excluded, resulting in a target list of 259 proteins.

A third set of target proteins was selected from a human cDNA expression library (hEx1), which was cloned in a bacterial expression vector. This library was screened for expression clones on high density arrays and by high-throughput protein expression and purification experiments 37383940. This identified 2,700 clones expressing soluble His-tag fusion proteins that could be purified by affinity chromatography 39. The cDNA inserts of these clones were sequenced and assigned to sequences of the Ensembl database 41. 141 proteins represented by clones of the expression library where selected as targets for structural analysis 39. These clones express soluble, full-length proteins, of which the three-dimensional structure was unknown.

The numbers of targets and success rates grouped by the type of target cDNA clone are summarised in Table 1.

Table 1

Origin of template cDNA clones Numbers of targets grouped by type of template cDNA clone

Target list

Number of targets

Number of successfully cloned cDNA

Number of proteins with soluble expression

Number of structures

1 – IMAGE clones

490

264

2 – cDNA consortium

259

185

3 – hEx1 library

141

all

890

537

139

Generation and characterisation of expression clones

We established a common cloning strategy that allows for easy shuttling of cDNA fragments between different E. coli and yeast vectors. We adopted a cloning system that adds only a minimal number of extra amino acids to the protein of interest and therefore decided to clone with restriction enzymes instead of using alternative systems, such as Invitrogen's Gateway system or ligation independent cloning 42.

The PSF has been working with more than a thousand target proteins to date. Suitable cDNA clones were selected and subcloned into the E. coli expression vectors pQTEV GenBank AY243506 and pQStrep2 AY02864216. These vectors provide for an N-terminal His-tag; pQStrep2 also encodes an C-terminal Strep-tag-II. Some proteins have also been expressed as GST fusion proteins using the vectors pGEX-4T2 or pGEX-6P1 (Amersham Biosciences).

Expression of protein coding genes from multiple transformants per target was tested under multiple conditions. Standardisation and automation was introduced to achieve this throughput. Expression clones were characterised by small scale protein synthesis at different temperatures, 37°C, 30°C and 25°C, in 1 ml volumes in deep-96-well microplates. Proteins were purified in parallel by a pipetting robot, as described previously 43. 10% of the purified protein eluate from a 1 ml culture was analysed by SDS-PAGE. For each protein expression experiment, the size of the expression product was recorded and the amount of protein was classified into four categories: none, weak, moderate and strong expression. This classification is arbitrary to a certain degree, however, we found it sufficient to select suitable clones for protein production scale-up.

1414 clones for 537 target proteins were successfully cloned in E. coli expression vectors, with 473, 191 and 94 target proteins corresponding to target lists one (IMAGE clones), two (DKFZ clones) and three (hEx1 clones), respectively. Clones for 139 different target proteins were found to be expressed in soluble form by E. coli. Figure 2 and Table 2 show the result of small scale expression and purification of these proteins. The yield varied significantly among different target proteins. The Additional file 1 psfClones.xml contains further details on the expression clones, such as vector, strain and helper plasmid for overexpression of rare tRNAs.

Figure 2

SDS-PAGE of purified human proteins

SDS-PAGE of purified human proteins. 15% SDS-PAGE (Coomassie-stained) of proteins expressed in small scale in E. coli and purified by automated immobilised metal chelate affinity chromatography as described in [43]. The identities of the purified proteins are indicated in Table 2. Protein expression was induced at the temperature that is optimal for the individual clone. These temperatures are listed in the supplementary file psfClones.xml. M: Molecular weight marker.

Table 2

PSF E. coli expression clones with soluble expression products. The table corresponds to the proteins shown in Figure 2. More detailed information is available in the supplementary XML file, Additional file 1.

NCBI Entrez gene ID

Gene symbol, name

Protein accession

RZPD clone ID

Sequence verified, non-silent mutations

Clone accession

Protein size [Da]

Protein gel, lane

ACAT2, acetyl-Coenzyme A acetyltransferase 2

AAM00223

PSFEp250C082

partly, none

44,177

D, 24

203

AK1, adenylate kinase 1

BAA78534

PSFEp250B112

yes, none

DQ000549

24,558

C, 21

689

BTF3, basic transcription factor 3

CAA37376

PSFEp758D0224

20,622

A, 27

830

CAPZA2, capping protein (actin filament) muscle Z-line, alpha 2

AAC60382

PSFEp758H1226

59,649

A, 4

1036

CDO1, cysteine dioxygenase, type I

BAA12872

PSFEp758D0124

yes, none

DQ000531

25,895

E, 16

1428

CRYM, crystallin, mu

AAC16914

PSFEp758B0810

yes, none

DQ000499

36,275

D, 4

1460

CSNK2B, casein kinase 2, beta polypeptide

CAA34379

PSFEp758H0422

26,209

B, 12

1606

DGKA, diacylglycerol kinase, alpha 80kDa

AAC34806

PSFEp758F0324

yes, none

DQ000529

16,408

A, 23

1627

DBN1, drebrin 1

AAH07567

PSFEp250C052

partly, none

74,354

C, 31

1635

DCTD, dCMP deaminase

AAC37579

PSFEp250B121

yes, none

DQ000535

22,938

E, 25

1937

EEF1G, eukaryotic translation elongation factor 1 gamma

AAH15813

PSFEp250H061

partly, none

53,045

C, 11

1974

EIF4A2, eukaryotic translation initiation factor 4A, isoform 2

AAH48105

PSFEp250H052

partly, none

49,301

D, 20

2963

GTF2F2, general transcription factor IIF, polypeptide 2, 30 kDa

CAA42419

PSFEp250B012

yes, none

DQ000547

31,304

C, 17

2992

GYG, glycogenin

AAB00114

PSFEp758H1122

yes, none

DQ000517

40,403

B, 4

3151

HMGN2, high-mobility group nucleosomal binding domain 2

AAA52678

PSFEp250E102

yes, none

DQ000559

12,314

D, 27

3312

HSPA8, heat shock 70 kDa protein 8

BAB18615

PSFEp250E022

partly, none

56,444

D, 31

3735

KARS, lysyl-tRNA synthetase

AAH04132

PSFEp250D032

partly, A116G

70,976

D, 16

3925

STMN1, stathmin 1/oncoprotein 18

CAC16020

PSFEp250F072

yes, none

DQ000556

20,225

D, 19

4043

LRPAP1, low density lipoprotein receptor-related protein associated protein 1

AAC67373

PSFEp250G031

yes, none

DQ000540

44,391

C, 22

4695

NDUFA2, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2

AAD27762

PSFEp250D091

13,843

C, 13

4698

NDUFA5, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5

AAD21526

PSFEp250A0510

16,381

A, 13

5184

PEPD, peptidase D

AAH28295

PSFEp250H122

yes, none

DQ000560

D, 29

5202

PFDN2, prefoldin 2

AAF17218

PSFEp250D112

yes, none

DQ000555

19,570

D, 17

5412

UBL3, ubiquitin-like 3

AAD02323

PSFEp758A0510

yes, none

DQ000501

15,657

5502

PPP1R1A, protein phosphatase 1, regulatory (inhibitor) subunit 1A

AAB02402

PSFEp758G0124

yes, none

DQ000526

21,862

A, 15

5716

PSMD10, proteasome (prosome, macropain) 26S subunit, non-ATPase, 10 (gankyrin)

AAH11960

PSFEp250A062

yes, none

DQ000544

27,351

C, 9

5717

PSMD11, proteasome (prosome, macropain) 26S subunit, non-ATPase, 11

AAH04430

PSFEp250B122

yes, none

DQ000548

50,390

C, 19

5877

RABIF, RAB interacting factor

AAB18264

PSFEp250C021

16,761

E, 23

6133

RPL9, ribosomal protein L9

BAA03401

PSFEp758E0124

yes, none

DQ000524

24,786

A, 28

6156

RPL30, ribosomal protein L30

CAA55820

PSFEp758A1113

yes, none

DQ000503

15,284

D, 6

6191

RPS4X, ribosomal protein S4, X-linked

BC007308

PSFEp758A0923

28,670

6342

SCP2, sterol carrier protein 2

AAA03559

PSFEp758C0723

yes, none

DQ000515

16,668

B, 1

6451

SH3BGRL, SH3 domain binding glutamic acid-rich protein like

AAC27445

PSFEp758C0713

15,274

D, 2

6728

SRP19, signal recognition particle 19 kDa

CAA31280

PSFEp758E0317

41,156

6888

TALDO1, transaldolase 1

AAB53943

PSFEp758B0711

partly, none

40,040

D, 1

6990

TCTE1L, t-complex-associated-testis-expressed 1-like

AAA57444

PSFEp758D0814

38,062

A, 3

6993

TCTEL1, t-complex-associated-testis-expressed 1-like 1

BAA09317

PSFEp758F1123

yes, none

DQ000521

13,719

A, 14

7001

PRDX2, peroxiredoxin 2

AAH03022

PSFEp250A042

yes, none

DQ000546

24,815

C, 30

7178

TPT1, tumor protein, translationally-controlled 1

CAA34200

PSFEp250G011

yes, none

DQ000539

22,518

C, 14

7247

TSN, translin

CAA55341

PSFEp250B111

yes, none

DQ000538

29,106

C, 12

7353

UFD1L, ubiquitin fusion degradation 1-like

N P_005650

PSFEp758G0123

yes, none

DQ000519

41,777

D, 9

7390

UROS, uroporphyrinogen III synthase

AAA60273

PSFEp758G0323

29,894

D, 11

7518

XRCC4, X-ray repair complementing defective repair in Chinese hamster cells 4

AAH16314

PSFEp250A033

partly, none

41,211

D, 26

7531

YWHAE, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon

AAH01440

PSFEp250H041

yes, A146G

DQ000541

32,097

C, 24

8125

ANP32A, acidic (leucine-rich) nuclear phosphoprotein 32 family, member A

AAB91548

PSFEp250D092

yes, none

DQ000553

31,509

D, 22

8214

DGCR6, DiGeorge syndrome critical region gene 6

CAA65339

PSFEp758G0324

yes, none

DQ000533

12,111

E, 19

8407

TAGLN2, transgelin 2

BAA04802

PSFEp250F062

yes, none

DQ000550

25,314

C, 27

8544

PIR, pirin

CAA69195

PSFEp758A0213

yes, none

DQ000498

34,613

C, 1

8575

PRKRA, protein kinase, interferon-inducible double stranded RNA dependent activator

CAB66550

PSFEp250D014

yes, none

DQ000578

37,328

E, 9

8677

STX10, syntaxin 10

AAC05087

PSFEp250D102

yes, none

DQ000552

25,369

D, 18

8724

SNX3, sorting nexin 3

AAC16040

PSFEp758A0323

yes, none

DQ000514

20,029

B, 15

8896

G10, maternal G10 transcript

AAH22821

PSFEp250F011

yes, A394G

DQ000536

19,766

C, 16

8926

SNURF, SNRPN upstream reading frame

AAD31391

PSFEp250A0610

11,334

C, 25

9049

AIP, aryl hydrocarbon receptor interacting protein

AAB59004

PSFEp758F1024

yes, none

DQ000528

40,589

A, 21

9158

FIBP, fibroblast growth factor (acidic) intracellular binding protein

CAG33030

PSFEp250B022

partly, T602C

44,803

C, 20

9168

TMSB10, thymosin, beta 10

AAB25225

PSFEp758A0124

yes, none

DQ000522

6,292

A, 18

9337

CNOT8, CCR4-NOT transcription complex, subunit 8

AAH17366

PSFEp758E0524

yes, none

DQ000523

36,354

A, 24

9453

GGPS1, geranylgeranyl diphosphate synthase 1

AAH05252

PSFEp250E052

partly, none

37,795

D, 23

9465

AKAP7, A kinase (PRKA) anchor protein 7

AAC39715

PSFEp758E0415

34,000

A, 1

9796

PHYHIP, phytanoyl-CoA hydroxylase interacting protein

BAA13402

PSFEp758C1124

yes, T806C

DQ000530

40,497

A, 29

10000

AKT3, v-akt murine thymoma viral oncogene homolog 3

CAB55977

PSFEp250B063

yes, none

56,516

E, 20

10063

COX17, COX17 homolog, cytochrome c oxidase assembly protein (yeast)

AAA98114

PSFEp758D0723

yes, none

DQ000516

8,182

B, 3

10094

ARPC3, actin related protein 2/3 complex, subunit 3

AAB64191

PSFEp758G0811

yes, none

DQ000507

23,047

D, 7

10169

SERF2, small EDRK-rich factor 2

AAC63516

PSFEp250A057

9,821

C, 23

10228

STX6, syntaxin 6

AAH09944

PSFEp758G0526

yes, none

DQ000509

32,100

B, 7

10247

HRSP12, heat-responsive protein 12 (14.5 kDa translational inhibitor protein, p14.5)

CAA64670

PSFEp758H0822

yes, none

DQ000518

15,760

D, 12

10290

APEG1, aortic preferentially expressed protein 1

AAH06346

PSFEp250B082

yes, none

DQ000543

15,614

E, 18

10539

TXNL2, thioredoxin-like 2

AAH05289

PSFEp250A022

partly, none

40,357

C, 26

10588

MTHFS, 5,10-methenyltetrahydrofolate synthetase

AAC41945

PSFEp758B0824

24,522

D, 15

10589

DRAP1, DR1-associated protein 1 (negative cofactor 2 alpha)

AAH10025

PSFEp250C042

yes, none

DQ000542

25,273

E, 3

10598

AHSA1, AHA1, activator of heat shock 90 kDa protein ATPase homolog 1 (yeast)

AAD09623

PSFEp250B091

41,199

C, 15

10606

PAICS, phosphoribosylaminoimidazole carboxylase

AAH19255

PSFEp758G0226

partly, none

50,005

A, 5

10842

C7orf16, chromosome 7 open reading frame 16

AAF03537

PSFEp758F0923

19,132

B, 2

10856

RUVBL2, RuvB-like 2 (E. coli)

AAH04531

PSFEp250C072

partly, none

54,083

D, 25

10912

GADD45G, growth arrest and DNA-damage-inducible, gamma

AAC83329

PSFEp758F0111

yes, none

DQ000504

19,621

C, 4

10933

MORF4L1, mortality factor 4 like 1

AAH22845

PSFEp250G051

partly, none

40,155

C, 10

10963

STIP1, stress-induced-phosphoprotein 1

AAA58682

PSFEp250A012

partly, A86C

65,566

C, 28

11140

CDC37, CDC37 cell division cycle 37 homolog (S. cerevisiae)

AAH08793

PSFEp250E042

partly, none

47,394

D, 28

11316

COPE, coatomer protein complex, subunit epsilon

AAH07250

PSFEp758G1126

yes, none

DQ000510

37,406

B, 9

11333

PDAP1, PDGFA associated protein 1

AAH07873

PSFEp250A073

yes, none

DQ000554

23,553

D, 30

11337

GABARAP, GABA(A) receptor-associated protein

AAD02337

PSFEp758H0922

15,185

D, 10

11344

PTK9L, PTK9L protein tyrosine kinase 9-like (A6-related protein)

N P_009215

PSFEp758A0127

yes, none

DQ000525

42,473

E, 13

22818

COPZ1, coatomer protein complex, subunit zeta 1

AAD34115

PSFEp758A0812

yes, none

DQ000497

22,698

D, 3

22919

MAPRE1, microtubule-associated protein, RP/EB family, member 1

CAB53072

PSFEp250C112

yes, none

DQ000557

32,923

D, 21

22931

RAB18, RAB18, member RAS oncogene family

CAB66668

PSFEp250C118

25,900

B, 27

23589

CARHSP1, calcium regulated heat stable protein 1

AAD25021

PSFEp778D072

40,934

25842

ASF1A, ASF1 anti-silencing function 1 homolog A (S. cerevisiae)

AAD34093

PSFEp758E1224

yes, none

DQ000532

25,891

E, 17

25843

PREI3, preimplantation protein 3

AAD34090

PSFEp758B0812

yes, none

DQ000500

28,532

C, 2

25996

DKFZP566E144, small fragment nuclease

AAD34109

PSFEp758G0423

yes, none

DQ000520

25,021

D, 13

26289

AK5, adenylate kinase 5

AAH12467

PSFEp250G042

yes, 3 bp insertion at 3' end

DQ000558

24,897

E, 14

26353

HSPB8, heat shock 22 kDa protein 8

CAB66870

PSFEp250G125

yes, none

DQ000565

24,527

B, 16

27095

TRAPPC3, trafficking protein particle complex 3

AAB96936

PSFEp758A0513

yes, none

DQ000502

22,774

C, 3

27249

C2orf25, chromosome 2 open reading frame 25

AAD20048

PSFEp758D1124

35,864

A, 22

27335

eIF3k, eukaryotic translation initiation factor 3 subunit 12

BAA76626

PSFEp758D0923

26,326

D, 8

27430

MAT2B, methionine adenosyltransferase II, beta

CAB66599

PSFEp250A114

yes, none

DQ000575

40,476

B, 21

28970

PTD012, PTD012

CAB66540

PSFEp250B014

yes, none

DQ000586

38,042

E, 24

29789

PTD004, GTP-binding protein PTD004

CAB66481

PSFEp250H073

yes, none

DQ000564

47,609

B, 18

30819

KCNIP2, Kv channel interacting protein 2

CAB66656

PSFEp250G064

yes, none

DQ000562

28,964

E, 5

51001

CGI-12, CGI-12

AAH12995

PSFEp250B092

yes, none

DQ000545

40,832

C, 8

51076

CUTC, cutC copper transporter homolog (E. coli), CGI-32

AAH21105

PSFEp758E1112

yes, none

DQ000505

31,811

C, 5

51078

THAP4, THAP domain containing 4

AAD27745

PSFEp250H116

21,550

C, 7

51155

HN1, hematological and neurological expressed 1

AAH01420

PSFEp758G0126

yes, 3 bp deletion at 5' end, G457A

DQ000508

18,937

C, 6

51160

VPS28, vacuolar protein sorting 28 (yeast)

AAF00499

PSFEp758C0323

yes, none

DQ000513

26,692

B, 14

51335

NEUGRIN, mesenchymal stem cell protein DSC92

CAD39160

PSFEp250B084

yes, none

DQ000566

27,342

B, 28

51397

COMMD10, COMM domain containing 10

AAD44489

PSFEp758H0811

yes, none

DQ000506

25,466

D, 5

51433

ANAPC5, anaphase promoting complex subunit 5

BAA76629

PSFEp758E0214

47,174

B, 8

51451

LCMT1, leucine carboxyl methyltransferase 1

AAH01214

PSFEp758H0126

partly, none

41,303

B, 11

51534

C6orf55, chromosome 6 open reading frame 55

AAF76210

PSFEp758H0426

partly, none

41,303

B, 13

51629

CGI-69, CGI-69

AAD34064

PSFEp758D0316

63,564

B, 6

51678

MPP6, membrane protein, palmitoylated 6

CAB66770

PSFEp250E023

yes, none

DQ000583

64,044

E, 27

54816

SUHW4, suppressor of hairy wing homolog 4 (Drosophila)

CAB66569

PSFEp250G074

yes, none

DQ000587

27,032

E, 22

55255

WDR41, WD repeat domain 41

CAD38853

PSFEp250E105

yes, none

DQ000563

26,313

55276

PGM2, phosphoglucomutase 2

CAB66640

PSFEp250B113

partly, none

71,239

B, 26

56681

SARA1, SAR1a gene homolog 1 (S. cerevisiae)

CAB66658

PSFEp250G115

yes, none

DQ000570

25,290

B, 17

56911

C21orf7, chromosome 21 open reading frame 7

CAD28500

PSFEp250B086

yes, none

DQ000571

19,032

E, 6

57019

LOC57019, cytokine induced apoptosis inhibitor 1

AAC24311

PSFEp758E0424

yes, none

DQ000534

36,506

E, 21

58485

TRAPPC1, trafficking protein particle complex 1

AAD44697

PSFEp250D071

yes, T350C

DQ000537

19,754

E, 1

64284

RAB17, RAB17, member RAS oncogene family

CAB66580

PSFEp250E095

yes, none

DQ000572

26,398

A, 6

79632

C6orf60, chromosome 6 open reading frame 60

CAB66701

PSFEp250B044

yes, none

DQ000568

34,217

B, 20

79666

PLEKHF2, pleckstrin homology domain containing, family F member 2

CAD39132

PSFEp250D054

yes, A271G

DQ000581

30,721

A, 9

79791

FBXO31, F-box protein 31

CAB66696

PSFEp250F085

yes, none

DQ000585

18,588

E, 12

80347

COASY, Coenzyme A synthase

AAF87955

PSFEp250A053

yes, none

DQ000551

33,147

C, 29

80895

ILKAP, integrin-linked kinase-associated serine/threonine phosphatase 2C

CAB66784

PSFEp250D083

yes, none

DQ000569

45,832

B, 19

81876

RAB1B, RAB1B, member RAS oncogene family

CAB66570

PSFEp250C128

25,094

A, 12

81889

DKFZP566J2046, fumarylacetoacetate hydrolase domain containing 1

CAB66654

PSFEp250B074

yes, none

DQ000567

27,766

B, 24

83538

DKFZP434H0115, hypothetical protein DKFZp434H0115

CAB66694

PSFEp250G093

partly, none

79,584

E, 11

83543

C9orf58, chromosome 9 open reading frame 58

CAB66501

PSFEp250B085

yes, none

DQ000573

19,990

B, 25

83667

SESN2, sestrin 2

CAB66486

PSFEp250F043

yes, none

DQ000576

57,420

E, 7

84072

NOHMA, HORMA domain containing protein

CAB66689

PSFEp250F083

yes, none

DQ000561

47,343

E, 4

84324

CIP29, cytokine induced protein 29 kDa

CAC37950

PSFEp758H1026

yes, none

DQ000512

26,594

A, 2

84457

PHYHIPL, phytanoyl-CoA hydroxylase interacting protein-like

CAD39006

PSFEp250A084

yes, none

DQ000579

45,425

E, 10

84557

MAP1LC3A, microtubule-associated protein 1 light chain 3 alpha

CAD38714

PSFEp250E106

yes, none

DQ000584

17,194

E, 26

91603

MGC20398, hypothetical protein MGC20398

BAB70992

PSFEp758H0526

yes, none

DQ000511

44,915

B, 10

94240

EPSTI1, epithelial stromal interaction 1 (breast)

CAD38599

PSFEp250C015

yes, none

DQ000580

25,527

A, 11

112611

RWDD2, RWD domain containing 2

CAB52345

PSFEp758A1024

partly, T709C

35,159

B, 5

118812

C10orf83, chromosome 10 open reading frame 83

CAD38849

PSFEp250H085

yes, none

DQ000574

19,158

B, 23

122060

FLJ30046, hypothetical protein FLJ30046

CAD38891

PSFEp250E015

yes, none

DQ000577

23,468

E, 8

136319

MTPN, myotrophin

CAD38909

PSFEp250D016

yes, none

DQ000582

15,817

A, 7

140856

C20orf79, chromosome 20 open reading frame 79

CAB56175

PSFEp758G0224

yes, G258T

DQ000527

20,585

A, 19

Biophysical properties of proteins which could be expressed in soluble form in E. coli were compared against all tested proteins. We found no significant correlation between expression success and either protein length or mean net charge (data not shown). However, when analysing the mean hydrophobicity, we found that hydrophobic proteins are less likely to be expressed in soluble form. Only one of 139 well expressed proteins has a mean hydrophobicity of more than 0.2, while 8% of the other proteins are above this value. This group of proteins does not contain transmembrane helices according to TMHMM, and therefore may represent peripheral membrane proteins with hydrophobic surface regions.

The E. coli expression clones of the PSF are publicly available from the RZPD German Resource Center 44. The Additional file 1 is an XML list of these clones. It can be viewed in a web browser (Figure 3). The Additional file contains, for each clone:

Figure 3

The supplementary XML Additional file 1 file displayed in a web browser

The supplementary XML Additional file 1 file displayed in a web browser.

• Gene ID and name,

• Accession number,

• Cloning details,

• Strain and vector,

• Expected sequence,

• Protein expression results,

• Sequence verification.

Solved structures

As a result of the target selection and cloning described in this paper, ten novel X-ray structures of human proteins were determined (Table 3). The structure of one protein, TRAPPC3/BET-3, was determined after protein expression in S. cerevisae, while the other proteins were produced in E. coli.

Table 3

Novel human protein structures The structures of full length proteins solved by the Protein Structure Factory.

NCBI Entrez gene ID

Name

GenBank protein accession

PDB ID

Reference

5716

Gankyrin

AAH11960

1QYM

[55]

10290

APEG1, aortic preferentially expressed protein 1

AAH06346

1U2H

81889

Fumarylacetoacetate hydrolase family member FLJ36880

CAB66654

1SAW

[56]

28970

PTD012

CAB66540

1XCR

10247

14.5 kDa translational inhibitor protein, p14.5

CAA64670

1ONI

[57]

27095

BET3, trafficking protein particle subunit

AAB96936

1SZ7

[58]

5184

Peptidase D

AAH28295

51076

CutC copper transporter homolog, CGI-32

AAH21105

122553

TPC6

CAI46185

2BJN

[59]

6449

Nicotinamide mononucleotide adenylyltransferase

NP_003012

1GZU

[60]

Discussion

We describe here the strategies and experiments of our structural genomics project on human proteins. In addition to the expression of full length proteins, the Protein Structure Factory has also studied protein domains by NMR spectroscopy, which has been described elsewhere 454647. Our selection of full length target proteins was mainly determined by the availability of full length cDNA clones. In addition, biophysical and bioinformatical criteria were applied, leading to a biased selection of target proteins from the human proteome. Therefore, we expect that the percentage of proteins that we could express and purify in soluble form, 18%, is higher than it would be in a randomly selected set. The low proportion of successfully expressed proteins indicates that E. coli is not the appropriate expression host for many full length human proteins. High throughput protein expression in alternative system such as yeast 91011 or insect cells/baculovirus 48 has been established and will lead to better success rates in future projects.

Generally, clones that did express a soluble protein were verified by DNA sequencing, while clones that did not express or expressed an insoluble product were usually not sequence verified. It cannot be ruled out that some of the unsuccessful clones contain sequence errors introduced during cloning. Since template cDNA clones of the IMAGE consortium with only partial sequence information were used for most cloning experiments, expression clones that were not sequence verified might represent splice variants or isoforms of the original target. The distribution of mean net charge and length was similar among successfully expressed and all proteins, while very hydrophobic proteins were generally not expressed well in our E. coli expression system.

Future efforts in structural genomics of mammalian proteins will benefit from a much better supply of full length cDNA clones. Clones prepared for protein expression by resource centres and commercial suppliers are becoming available now. With such resources, alternative target selection strategies will become feasible that will not be restricted by the availability of cDNA clones. Instead, all potential target proteins, including splice variants, could be clustered by similarity and the most suitable members of each cluster could be selected by appropriate criteria as outlined in the Background section.

In our approach, we have excluded certain types of proteins such as membrane proteins and very large proteins. A structural genomics approach that includes membrane proteins would require standard protocols to optimise expression conditions and detergents 49. The best strategy to study large proteins is to divide them into domains and smaller regions. However, such smaller constructs usually have to be designed manually.

All clones listed in the supplementary file (Additional file 1) and Table 2 are available to the research community. Thereby we hope to facilitate further functional characterisation of this set of human proteins.

Materials and Methods

Cloning with restriction enzymes

cDNA inserts were amplified by PCR primers carrying tails with BamHI and NotI sites and cloned into the respective sites of one of the expression vectors pQTEV, pQStrep2 or pGEX-6p1. This had the drawback that the restriction sites chosen for cloning might occur in the insert. In such cases, compatible overhangs were produced by alternative enzymes or by the hetero-stagger cloning method 50. Alternative enzymes are BglII for BamHI and the type IIs enzymes BpiI, Eco31I, Esp3I, which can replace both BamHI and NotI. Type IIs enzymes cut outside their recognition sequence and can produce arbitrary overhangs.

PCR Primer design

PCR primers with tails carrying restriction enzyme cleavage sites were designed automatically by a Perl program. The primer design program adjusts the length of the primers to achieve a melting temperature close to a common default. Then, restriction enzymes that do not cut within the respective cDNA sequence are selected by the program and restriction enzyme sites are attached to the primer sequences. Finally, since restriction enzymes do not cut well at the very end of a DNA molecule, an additional short nucleotide tail is automatically attached to the primers. The sequence of this tail is optimised to minimise formation of secondary structure, hairpins or dimerisation. A Java version of the primer design software, 'ORFprimer', is publicly available 51.

Automated high-throughput cloning, protein expression and purification

PCR primers and cDNA clones were delivered in 96-well microplate format. Upon delivery, plates with PCR primers and template clones were reformatted to obtain corresponding plate positions by a Zinsser Speedy pipetting robot. The PCR master mix (Roche Expand) and cDNA primers (10 μM stocks) were pipetted into a PCR microplate with a multichannel pipet. Template clone bacteria were added with a 96-pin steel replicating device from overnight cultures in microtitre plates. PCR product size and yield was determined by agarose gel electrophoresis and the software Phoretix 1D Quantifier (Nonlinear dynamics). PCR products were purified with magnetic beads on the pipetting robot with a system that has been developed at the Max Planck Institute of Molecular Genetics in collaboration with Bruker Daltonics (Bruker genopure kit). The correct restriction enzyme master mixes were automatically added and the digested fragments were purified again, analysed by agarose gel electrophoresis and quantified. The robot then adjusted DNA concentrations to a common default by dilution. Ligations were set up manually with a multichannel pipet, and SCS1 E. coli cells carrying pRARE were transformed in a PCR microplate by chemical transformation on a PCR machine 52. Transformed cells were manually plated on individual agar plates. Four clones were picked per transformation and were checked by PCR using vector primers. E. coli expression clones were ready for protein expression at this stage.

Primer sequences and template clones for cloning of the target cDNAs are listed in the supplementary XML file, Additional file 1.

The characterisation of expression clones by parallel expression and protein purification is described in reference 43.

Sequence analysis

Sequence analysis software was run with default settings unless indicated otherwise. The mean charge of a protein was calculated as the difference of the number of positive and negatively charged amino acids (Lys, Arg and Glu, Asp, respectively) divided by the protein length. The mean hydrophobicity was calculated with the Kyte and Doolittle hydropathy index 53, obtained from the EMBOSS package 54. The index values were added up for a given protein and divided by the protein length.

Authors' contributions

• Study design and coordination: KB, HL, UHe

• Bioinformatics: UHa, BS, JS, PB, VS, KB

• E. coli cloning: VS, KB

• E. coli protein work: CS, KB

• Manuscript, figures, XML file preparation: KB

Additional File 1

A ZIP archive that contains the XML list psfClones.xml of PSF clones available at the RZPD and the XSL stylesheet psfToHtml.xsl to display the XML file in a web browser. The two files should be extracted from the ZIP archive into a local directory. Then psfClones.xml can be opened with a current web browser like Mozilla or Internet Explorer. 1414 Clones for 537 target proteins are described in the XML file. It lists the gene, cloning details, expected sequence, protein expression results and the degree of sequence verification for each clone.

Click here for file

Acknowledgements

We wish to thank Ulf Lenski for help with sequence analysis and Janett Tischer for technical assistance. This work was funded by the German Federal Ministry of Education and Research (BMBF) through the Leitprojektverbund Proteinstrukturfabrik and the National Genome Network NGFN (FKZ 01GR0471, 01GR0472) and with support by the Fonds der Chemischen Industrie to U.H. Support by the Berlin Senate and the European Fund for Regional Development (EFRE) is also gratefully acknowledged.

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