Production and Purification of Recombinant Membrane Proteins

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Production and Purification of Recombinant Membrane Proteins ETANA PAD AN Microbial and Molecular Ecology Institute of Life Sciences Hebrew University of Jerusalem 91904 Jerusalem, Israel

CAROLA HUNTE Molekulare Membranbiologie Max-Planck-Institut fur Biophysik 60528 Frankfurt am Main, Germany

HELMUT REILANDER Aventis Pharma, Industriepark Hoechst 65926 Frankfurt am Main, Germany

I. INTRODUCTION The investigation of membrane proteins has become an active area in biochemical and biomedical research. For many experimental approaches, large quantities as well as high concentration of protein are needed. Structural analysis by NMR or x-ray crystallography requires, for example, several milligrams of functional protein in concentrated solutions ('^0.25 mM). However, membrane proteins often exist in their native membranes at low abundance. NhaA, the sodium/proton antiporter of Escherichia coli, comprises for instance less than 0.2% of the total membrane proteins (Taglicht et ah, 1991). Therefore, strong overexpression is needed to attain a high cellular concentration of the protein of interest. While for many soluble proteins

Membrane Protein Purification and Crystallization 2/e: A Practical Guide. Copyright 2003, Elsevier Science (USA) All rights of reproduction in any form reserved.

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suitable overexpression systems are used routinely, high-level production of membrane proteins is still challenging. Overexpression of both homologous as well as heterologous proteins is often deleterious to the membrane and thus to the growth of the host organism (Miroux and Walker, 1996). A variety of expression systems for membrane proteins in bacteria, as well as in eukaryotic cells, has been developed to overcome these difficulties. Crucial steps in the production of membrane-bound proteins are not restricted to transcription and translation but include the insertion into and the folding of the protein within the membrane. The latter processes are often the rate-limiting steps in production. If correct insertion and folding fail, large quantities of inactive and partly aggregated protein are produced and accumulate within the cell. Although membrane protein insertion and folding follows general principles (Popot, 1993; Popot et ah, 1994), differences in the mechanisms between the prokaryotic and the eukaryotic systems are evident (Dobberstein, 1994). The insertion of a prokaryotic membrane protein is a co-translational process (deGier and Lurirink, 2001). The co-translational insertion of a protein into the membrane involves the signal recognition particle and its receptor (MacFarlane and Mueller, 1995; Seluanov and Bibi, 1997). A membrane potential is needed for correct membrane protein insertion in E. coli, a requisite not known for eukaryotic systems (Bassilana and Gwidzek, 1996; Wickner and Lodish, 1985). To date it is assumed that eukaryotic membrane proteins are cotranslationally inserted into the ER membrane, where they subsequently fold into their final three-dimensional conformation (Borel and Simon, 1996; Do et ah, 1996; Wessel and Spiess, 1988). In many cases, this process is supported by specialized folding proteins, so-called chaperones, like the BiP ("heavy chain binding protein" a member of the Hsp 70-heat shock protein family), the disulfide-isomerase and/or calnexin (Bergeron et al,, 1994; Hartl et ah, 1994; Helenius et al., 1992). In general, molecular chaperones prevent false intraand/or intermolecular interactions during the complicated folding process of proteins (Bergeron et al, 1994; Hartl et al, 1994; Gething and Sambrook, 1992). In only a few cases, there is exact knowledge of when and/or which chaperones are needed for correct folding of membrane proteins. NinaA, which acts as a peptidyl prolyl cis/trans isomerase, has been found to be absolutely essential for production of functional rhodopsin 1 and 2 in the eye of Drosophila melanogaster (Baker et al, 1994; Ondek et al 1992). In the absence of the ninaA protein, the rhodopsins aggregate and accumulate in the ER (Baker et al, 1994). If such specialized chaperones are needed for the correct production and, in particular, for the correct folding of recombinant membrane proteins, heterologous production appears to be very difficult to achieve. Here, the coproduction of specific chaperones might be a solution, which could finally lead to the design of a specialized host cell system (Lenhard and Reilander, 1997).

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Posttranslational modifications should be considered when producing a recombinant protein. Almost all eukaryotic membrane proteins that have been detected in the plasma membrane are glycosylated. Glycosylation of a protein might be important for its function and/or required for its correct folding, for the assembly of subunits, and also as a signal to guide the protein to the proper subcellular location (Geisow, 1992). Additional posttranslational modifications—for example, phosphorylation or palmitylation of receptors—are of functional importance. If posttranslational modifications are an absolute prerequisite for the proper function of a membrane protein, heterologous production can only take place in an expression system that is capable of performing these kind of modifications. Then, prokaryotic expression systems have to be excluded, and production of the respective protein has to be performed in an eukaryotic system (Geisse et ah, 1996). High-level production of active eukaryotic membrane proteins in E. coli, the most widely used bacteria, is still a challenge. This heterologous expression system often results in low yield, or the proteins tend to form insoluble inclusion bodies with inactive protein. Inclusion bodies also form during both homologous as well as heterologous overexpression of prokaryotic proteins in prokaryotic systems. Refolding of functional proteins from inclusion bodies has been described for very few membrane proteins (see Section II,C). In comparison to natural protein sources, expression systems for recombinant proteins offer an enormous potential for selective alteration of the protein structure to improve its properties for various aims. Removal of internal proteolysis or glycosylation sites will reduce heterogeneity of the product. Furthermore, protein or peptide fusions can be added to the protein of interest to facilitate its detection and purification. Expression constructs can be tailored to increase solubility, stability, and yield of the protein.

11. PROKARYOTIC EXPRESSION SYSTEMS FOR OVERPRODUCTION OF MEMBRANE PROTEINS FOR STRUCTURAL STUDIES Various strategies have been exploited in bacteria to achieve overexpression of membrane proteins: (1) use of strong promoters along with the proper transcription initiation and translation signals for overexpression; (2) exploitation of tightly regulated promoters to attain high biomass of bacteria before induction, followed by a limited induction time to obtain high amounts of the protein before the death of the bacteria; (3) use of leaky promoters without induction combined with low temperature to allow sufficient time for insertion and folding processes (Grisshammer and Tate, 1995); and (4) refolding proteins from inclusion bodies.

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The advantages in bacterial expression systems are as follows. 1. Bacterial protein expression is cheaper and faster than eukaryotic systems. 2. Overexpression is obtained without any posttranslational modification that can introduce heterogeneity, potentially harmful for crystallization. However, for proteins that require posttranslational modifications for proper folding and activity, eukaryotic expression systems are needed (see Section III). 3. Escherichia coli is the most frequently used bacteria for protein production due to its highly developed molecular genetics. Furthermore, advancing the understanding of the fundamental aspects of E. coli physiology—that is, protein folding, protein excretion, transcription, and translation—will continue to fuel progress in optimizing this organism for protein expression. This section is limited to expression systems developed in £. coli.

A, E. coli Inducible Promoters Used for Overexpression Plasmid-based inducible promoter systems have been constructed in E. coli using bacterial, phage, and chimeric promoters (Table I). One of the most widely used expression systems in E. coli is based on the T7 RNA polymerase (Studier et ah, 1990). The coding sequence of the enzyme is cloned into the E. coli chromosome downstream of an inducible promoter—for example, the IPTG inducible lac promoter from the excision-deficient A-lysogen DE3 (often used is the UV5 lac promoter that is insensitive to catabolite repression, [see Grossman et ah, 1998)]. The target gene is introduced into this bacteria on a plasmid positioned downstream to the T7 promoter. Therefore, target gene expression via the T7 RNA polymerase is inducible by IPTG. Other variations on this theme exist (for examples see Table I and Durbin, 1999). A thorough study of overexpression of several membrane proteins using this system and the E. coli strain BL21 (DE3) showed cell-toxicity caused by overexpression (Miroux and Walker, 1996). Mutants, C41 (DE3), C43 (DE3), surviving the toxicity were selected and shown to overproduce the proteins without toxic effect. Although neither the mechanism of toxicity nor the relevant mutations are known, the mutations are claimed to reside in the T7 RNA polymerase and to balance its activity with respect to translation. The procedure used for selection of these mutants could be employed to tailor expression hosts in order to overcome toxic effect caused by the T7 RNA polymerase based overexpression systems. The RNA polymerase of phage T7 is inhibited by the phage lysozyme (review by Durbin, 1999). Therefore, cotransformation of cells with one plasmid encoding lysozyme (pLysS and pLysE) and a second plasmid encoding the target protein has been advocated to suppress basal expression toxicity.

CHAPTER 3

TABLE I

Controlled Expression Syst ems Used in E. coli

Promoter used for^ overexpression of the target gene cloned on plasmid

Regulatory manifold^

17 RNA polymerase

Induction

Level

Ref.

JpjQd

Very high XlOO

[1]

Temperature shift to 42°C

Moderately high

PL(^)

T7 RNA polymerase expressed from the lac promoter or derivatives in the presence of lacl^ (overexpressed repressor) The PL promoter in the presence of a)cV'S57 temperature sensitive repression The proU promoter Mutated 77 RNA polymerase in strain BL21 (DE3)'^; balancing transcription translation Lad, lacl^ repression (/l)cP^857 repression

araBAD

AraC repressor

Temperature shift to 42°C L-arabinose

tet

Tet repressor

Anhydrotetracycline

lac, tac, trc

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Production and Purification of Recombinant Membrane Proteins

NaCl IPTG

[2] [3]

IPTG

Moderately high Moderately high Fine-tuning (but see (5) and below) Very high

[4]

[5] [6]

''In addition to the promoter, other regulatory signals can be manipulated; DNA sequences important for transcription a n d / o r translation. ''Can be on the chromosome or plasmid. ^The site of the mutation has not yet been proven. The strains were successfully used to overexpress membrane proteins (Besette et ah, 1999; Miroux and Walker, 1996). '^IPTG, isopropyl jS-D-thiogalactoside. [1] For review, see Stevens, 2000; [2] Bhandri and Gowri-shankar, 1997; [3] Mulroony and Washell, 2000; Fiermont et al, 2001; [4] Brosius, 1999; [5] Guzman et a/., 1995; [6] Lutz and Bujard, 1997.

Various derivatives of the lac promoters are used for overexpression in addition to the native lacV (Table I). These are hybrids constructed from the — 35 consensus sequence of the trp promoter and the —10 of the lacV spaced by 16 bp in the case of tacV or by 17 bp in the case of trcV, Expression plasmid systems based on the lac promoter are notoriously leaky. Repression is often incomplete due to a combination of plasmid copy number effects and the absence of secondary operators required for the full range of gene control in

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the natural lac operon (review by Durbin, 1999). This problem is often overcome by the use of overexpressed repressor LacI^ or Lacl^l and/or the addition of an operator. Background expression levels should be lower in systems based on positive rather than negative control of expression. Recently, expression plasmids based on the araBAD promoter (FaraSAD) have been constructed (Guzman et al, 1995). Because the ara system can be induced by arabinose and is repressed by either catabolite repression in the presence of glucose or competitive binding of the anti-inducer fucose, these plasmids have very low background levels of expression. In addition, gene expression can be turned on and off rapidly by changing the sugars in the medium. Ideally, one would like to be able to modulate the level of gene expression. Guzman et al. (1995) presented evidence that the pBAD vectors are suitable to express at intermediate levels. However, intermediate levels of gene expression in cultures do not always mean that gene expression is uniform with respect to individual cells. In extreme cases, either every cell in the culture makes the same percentage of the fully induced level, which is observed in the culture, or the culture consists of a mixture of fully induced and completely noninduced cells. In the latter case, the intermediate expression levels observed in the culture reflect the proportion of cells that are fully induced, rather than intermediate expression in any individual cell. The latter alternative was found correct using Aequorea victoria green fluorescent protein (GFP) as a reporter (Siegele and Hu, 1997). Expression in cultures grown at low concentrations of arabinose is bimodal, consistent with an autocatalytic mechanism for induction of transcription of ParaSAD due to active uptake of the inducer. This phenomenon is analogous to the autocatalytic induction of the lac operon (Novick and Weiner, 1957). Expression from the lac promoter can be modulated using different concentrations of the membrane-permeable inducer IPTG in a lacY~ strain, where in the absence of an inducible transport system the level of expression in individual cells in the population is relatively homogeneous. Plasmids with promoters controlled by lac repressor have been used to modulate expression of genes over a 20- to 250-fold range (Hashemzadeh-Bonehi et al., 1998; Malony and Rotman, 1973; Prinz et al., 1997). The importance of performing such experiments with other systems should be emphasized. A recent review (Baneyx, 1999) describes the potential use for protein overexpression of other E. coli promoters, upstream and downstream sequences involved in transcription or translation, and the recent advancements in manipulating the copy number of the target gene (Baneyx, 1999).

B. Optimizing Expression Levels in E. coli Having a good nontoxic expression plasmid does not automatically guarantee that the protein is produced in large quantities. To achieve this goal

CHAPTER 3 Production and Purification of Recombinant Membrane Proteins TABLE II

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E, coli Genotypes Relevant for Overexpression Relevant genotype

Relevant phenotype

endA Ion ompT argJJ ileY leuU proL extra copies of genes encoding tRNAs frequently limiting translation trxB, gor

Endonucleasel" Lon cytoplasmic protease" OmpT outer membrane protease" ArgU^, IleU"^, leuU"^, for AT-rich genomes ArgU^ and ProL"^ for GC-rich genomes"

dsbC (engineered in a trxB cytoplasm) met lacY lac operator

Thioredoxin reductase ", glutathion reductase " allow disulfide bond formation in the cytoplasm [1] Disulfide bond isomerase" [2] Methionine auxotrophe allows high specific activity labelling with ^^S methionine or selenomethionine for crystallography Lac permease" allows fine tuning with lac derived promoters [3] Addition for tight control with lac repressor

"For codon usage problems, it is also possible to systematically mutate the offending codons to synonymous codons of E. coli. [1] Prinz et al, 1997; [2] Bessette et al, 1999; [3] Hashemzadeh-Bonehi et al, 1998.

it may be necessary to prevent mRNA degradation, to remove undesirable features in the coding sequence that impede translation, to prevent proteolytic degradation, or to assist in protein folding. Hence, in every case of overexpression, empirical trials need to be performed for optimization, altering expression conditions, and observing the yield, solubility, and stability of the recombinant protein. The conditions that should be optimized are temperature, inducer concentrations, and media. In some cases, supplements of salts or trace elements (Zn^"^, Cu^"^, Ca^"^, Fe^"^, Fe^"^) and potential cofactors or prosthetic groups (e.g., heme, FAD, FMN, tetra hydro-biopterine) are needed. Host strain genotype is also important for obtaining optimal expression levels (Table II). Many E. coli strains have been developed for this purpose. The properties manipulated are high competence (usually patented); End A" for cloning expression constructs (endonuclease I is an enzyme that rapidly degrades plasmid DNA); Lon protease"; or OmpT protease" (these proteases can degrade recombinant proteins). Efficient production of heterologous proteins in E. coli is frequently Umited by the rarity of certain tRNAs that are abundant in the organisms from vvrhich the heterologous proteins are derived. Forced high-level expression of heterologous proteins can deplete the pool of rare tRNAs and stall translation. Strains are engineered to contain extra copies of genes that encode the tRNAs that most frequently limit translation of heterologous protein in E. coli.

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Availability of tRNAs allows high-level expression of many heterologous recombinant genes. These can be of high significance for genes derived from AT-rich or GC-rich genomes. Factors involved in protein folding are recruited now to improve overexpression and combat the problem of inclusion body formation. These include chaperones and peptidyl propyl cis/trans isomerases (for further reading, see Baneyx, 1999). Disulfide bonds do not form in the cytoplasm of wild-type E. coli strains, since this environment is reducing, trx^ encodes thioredoxin reductase, a protein responsible for the reduction of oxidized thioredoxins; gov encodes glutathion reductase. Deletion of these genes appears to facilitate accumulation of proteins containing structural disulfide bonds in the cytoplasm (Prinz et al., 1997; Venturi ei al., 2002). Another option is to direct the disulfide bond containing domain to the periplasm. There the environment is oxidizing and contains enzymes catalyzing the formation and rearrangement of disulfide bonds (Cornelis, 2000).

C. Inclusion Bodies and Refolding Overexpression of native proteins, but especially of heterologous recombinant proteins, leads to the formation of insoluble protein aggregates in the cytoplasm known as inclusion bodies. Current estimates are that 15-20% of human gene constructs expressed in E. coli are soluble, 20-40% form inclusion bodies, and the remainder do not express significantly or are degraded (for a recent review, see Rudolph and Lilie, 1996). Inclusion bodies are formed in both eukaryotes and prokaryotes, and there is no direct correlation between the propensity of their formation and intrinsic properties of the protein. For proteins containing disulfide bonds, inclusion body formation can be anticipated because the reducing cytosol inhibits disulfide bond formation (Hauke et al, 1998). Two strategies exist for dealing with inclusion bodies: (1) Minimize or avoid them by increasing the fraction of the protein that remains soluble. This involves modifications in the expression system (both host and vector) (review in Rudolph and Lilie, 1996; Hauke et al, 1998; Stevens, 2000); induction at lower temperatures; lowering the concentration of inducing agent; altering the media composition (e.g., adding sucrose or polyols); coexpressing chaperones and other in vivo folding enhancers; expressing the protein as a fusion with a "solubilizing partner" such as GST, MBP, thioredoxin, or NusA (see below). (2) Since inclusion bodies contain almost exclusively aggregates of the overexpressed protein, driving protein expression to inclusion body formation can be advantageous. Proteins in inclusion bodies are generally protected from proteolytic degradation. The use of inclusion body routes is especially attractive for the production of proteins that are toxic to host cells. Properly folded proteins can be produced from these inclusion bodies using a variety of

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solubilization and in vitro refolding schemes (for review, see Hauke et ah, 1998). Partial success at in vitro refolding has been obtained with hydrophobic interaction chromatography. Unfortunately, refolding screens must still be developed, especially as different proteins have highly variable refolding efficiencies. During generic refolding processes, severe heterogeneity problems can arise owing to the formation of multiple refolded species and aggregates. Hence, efforts aimed at recombinant protein refolding should probe as many factors as possible to maximize the probability of obtaining properly folded protein in its native conformation. Very few membrane proteins were produced by overexpression in E. coli as inclusion bodies and successfully refolded. These include two transport proteins from the mitochondrial inner membrane that appear ready for crystallization attempts (Fiermonte et ah, 1993; 2000) and a G-protein coupled receptor (Echtay et ah, 2000; Kiefer et ah, 1996,1999, 2000). Immobilization on nickel chelating chromatography material was succesfuUy used for refolding Toc75, the translocase of the chloroplast outer envelope, and LHC2, a pigment binding oligomeric assembly (Rogl et ah, 1998).

III. EUKARYOTIC EXPRESSION SYSTEMS FOR OVERPRODUCTION OF MEMBRANE PROTEINS Many eukaryotic expression systems have been tested for heterologous production of membrane proteins, including several single cell yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), insect cells, and mammalian cells (for reviews, see Grisshammer and Tate, 1995; Reilander et ah, 1999; Schertler, 1992). For the investigation of structure/function relationship of a protein by site-directed mutagenesis, transient expression of the modified protein in mammalian cell lines is the method of choice. It allows fast evaluation of the different altered protein isoforms. The most commonly used cell lines are those from Chinese hamster ovary (CHO), baby hamster kidney (BHK), human embryonic kidney (HEK) 293, and NIH3T3. Transient and stably transfected mammalian cell lines are also well suited for functional investigation of different receptor subtypes as well as for establishment of search systems for new receptor ligands by "high-throughput-screening" (HTS) a n d / o r ultra-HTS (UHTS) approaches (Stratowa et ah, 1995). Due to the presence of intact signalling pathways in most of the mammalian cell lines, analysis of signal transduction of receptors can easily be performed (Wilson et ah, 1999). However, for large-scale production and purification of membrane proteins, mammalian cell systems are less suited. These aims can be achieved by the Pichia pastoris system and the insect cell/baculovirus system, which will be introduced in more detail in this section.

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A. Yeast-Based Expression Systems Yeast-based expression systems are very attractive for heterologous production of membrane proteins. They are easy to manipulate and grow to high cell yield at low cost. Yeasts have been used in fermentation and food industry for decades and have been proven safe. Contrary to E. coli, yeasts have the potential to perform posttranslational modifications, which in some cases are needed for production of functional protein (Buckholz, 1993; Cereghino and Cregg, 1999, 2000; GeUisen and HoUenberg, 1997). Besides the classical expression system of S. cerevisiae, several other yeast systems have recently become popular. These include Schizosaccharomyces pombe, P. pastoris, P. methanolica, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, or Candida utilis. However, only S. cerevisiae, Sz. pombe, and P. pastoris, have been tested for production of membrane proteins [for example: G-protein coupled receptors (GPCRs); see Reilander et ah, 1999 and a multidrug transporter (MRPl) (Cai et al., 1991)]. S. cerevisiae is the best characterized eukaryotic organism in terms of molecular genetics, cell biology, and physiology. Its complete genome sequence has been recently published (Goffeau et ah, 1996), and there are many ongoing studies to elucidate the function of newly discovered genes. A multitude of different strains including protease-deficient strains as well as a variety of expression vectors comprising yeast episomal plasmids (Yeps) and yeast integrating plasmids (Yips) are commercially available and allow easy genetic manipulations. The internal GPCR signal pathway, for example, has been modified and adapted to create a host for high-throughput screening of GPCRs (Pausch, 1997). However, S. cerevisiae has some limitations as a host: (1). It does not produce high cell densities, (2) during fermentation, plasmids, especially Yeps, may be unstable, and (3) glycoproteins may be incorrectly glycosylated—for example, hyperglycosylated. The fisson yeast Sz. pombe is phylogenetically as related to S. cerevisiae as it is to mammalian cells. It has many properties in common with higher eukaryotic organisms (Giga-Hama and Kumagai, 1997). A range of different expression vectors for Sz. pombe have recently become commercially available, some of which are based on the thiamine-repressible nmtl promoter {nmtl, no message in thiamine) (Maundrell, 1993). However, the biotechnological application of this yeast lags behind that of S. cerevisiae and P. pastoris. 1. Pichia pastoris as an Expression System The use of yeast as an expression system commenced when Phillips Petroleum converted the methylotrophic yeast P. pastoris into a producer of recombinant proteins. P. pastoris is one of about a dozen yeast species that can use methanol as its sole energy and carbon source. The metabolic pathway

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needed for methanol consumption involves a set of different enzymes where alcohol oxidase I (AOXl) catalyzes the first step. The expression of the AOXl gene is tightly regulated and specifically induced by methanol. More than 30% of the cell protein is AOXl when the cells grow on methanol in a fermenter. The first steps of the methanol utilizing pathway take place in specialized organelles, the peroxisomes. This compartmentalisation prevents possible oxidative damage to the cell from the production of hydrogen peroxide. In most cases, expression of foreign genes in P. pastoris was accomplished by using the strongly regulated promoter derived from the AOXl gene (Higgins and Cregg, 1998). Other vectors that use the promoter of the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) gene (Waterham et ah, 1997) were constructed. Strains and vectors are commercially available (Invitrogen). P. pastoris is always used in combination with vectors for genomic integration. After transformation and integration of the linearized recombinant vectors, the resulting recombinant yeast clones are extremely stable even when induced. The techniques for molecular genetic manipulation of P. pastoris are very similar to those of S. cerevisiae. Additionally, P. pastoris has a strong preference for respiratory growth, a property that facilitates growth to extremely high cell densities of up to 500 g per liter culture medium. To date, more than 100 different proteins have been successfully produced in P. pastoris, including membrane-bound receptors (GPCRs) and transporters (Cereghino and Cregg, 1999, 2000). Four different methods are available for the transformation of P. pastoris. Although more laborious than the others but best characterized is the spheroblast method that produces transformants at high frequency. The other three methods rely on intact untreated cells. The most common whole-cell method of transformation is based on electroporation, which results in almost as many recombinants as the spheroblast method. The other two transformation procedures have the advantage that they do not need special equipment. Both of these methods, the PEG-transformation as well as the LiCl-method, result in the generation of an adequate number of transformants (Cregg and Russell, 1998). To date, all P. pastoris strains available are derivatives of the NRRL-Y11430 strain (Table III). To allow for selection of transformants that carry a vector containing the histidinol dehydrogenase gene (HIS4), most strains bear a mutation in HIS4. These strains are able to grow on complex media, but for their growth in minimal medium, histidine has to be supplemented. The most commonly used host for transformation is strain GS115, which posesses a wild type AOXl and grows rapidly on methanol (Muf^ phenotype). In contrast, the strain KM71 has a reduced growth rate on methanol (Mut^, methanol utilization slow), since the chromosomal AOXl gene was largely deleted and replaced by the S. cerevisiae ARG4 gene. The slow growth of this strain on

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TABLE III

P. pastoris Strains Strain GS115 X33

KM71

KM71H

SMD1168 SMD1168H

SMD1163

Genotype (application) his4 (selection of vectors containing HIS4) wild type (selection of vectors containing the zeocin resistance gene) aoxl::ARG4 his4 arg4 (selection of vectors containing HIS4 resulting in clones with Mut^ phenotype) aoxl::ARG4 arg4 (selection of vectors containing the zeocin resistance gene resulting in clones with Mut^ phenotype) Apep4his4 (selection of vectors containing HIS4) Apep4 (selection of vectors containing the zeocin resistance gene) Apep4 his4 Aprbl (selection of vectors containing HIS4)

Phenotype

Ref.

Mut+His"

[1]

wild type

[2]

Mut^His"

[31

Mut'His + protease-deficient

[2]

Mut+His"; protease-deficient Muf^His^; protease-deficient

[4]

Mut+His"; protease-deficient

[5]

[2]

Adapted and expanded according to Higgins and Cregg (1998). [11 Cregg et al, 1985; [21 Invitrogen; [3] Cregg and Madden, 1987; [4] White et al, 1995; [5] Gleeson et al, 1998.

methanol relies on the activity of a second alcohol oxidase gene, AOX2, which is present in the P. pastoris genome. A0X2 has an explicitly lower activity compared to AOXl. With many expression vectors it is possible to replace the AOXl gene in the GS115-related strains with an expression casette by homologous recombination resulting in a Mut^ phenotype. Proteolysis is a problem during production of recombinant proteins, especially membrane proteins (Weiss et al, 1995). The vacuolar proteases play a major role in this proteolysis. Therefore, the use of the protease-deficient strains SMD1168 or SMD1163 (Table III) is highly recommended. The PEP4 gene encodes for the vacuolar proteinase A, which is required for the activation of several other vacuolar proteases. The PRBl gene codes for proteinase B. SMD1163, a strain bearing the double mutation pep4 prbl, shows a significant reduction in protease activities and, therefore, provides an optimal environment for production of sensitive proteins. The strains X33, KM71H, and SMD1168H (Table III), which all bear no mutation in the HIS4 gene, are designed for transformation with expression vectors conferring resistance either to Zeocin or Blasticidin.

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The most commonly used vectors for heterologous production of proteins in P. pastoris are shuttle vectors (Table IV). Some vectors bear the wild-type HIS4 gene and the bacterial ampicillin resistance gene as selectable markers. The HIS4-based vectors are in general large (up to 10 kb), and the multiple cloning site is not very convenient. The AOXl promoter region present on these vectors is followed by a multiple cloning site suited for insertion of the foreign gene followed by the AOXl transcriptional termination sequence. The 3' flanking region of the AOXl gene contains the HIS4 selection marker, which is needed for screening the integration of the expression cassette at the AOXl locus by gene replacement. Furthermore, it contains sequences required for replication and propagation of the vectors in E. coli (for example, the pPIC9 vector). Generation of recombinant strains bearing multicopy integrations of the expression plasmid in the genome has been shown to result in an increased production of heterologous protein via a gene dosage effect. Special vectors that carry a second selection marker in addition to HIS4 were designed for this application. They contain the E. coli kanamycin gene of transposon 903 inserted between HIS4 and the 3' flanking region of AOXl (for example, the pPIC9K vector). This marker allows identification of recombinant clones bearing multicopy integration of the expression cassettes by screening for increased resistance to geneticin (G418). In the first step Pichia transformants are selected on histidine-deficient medium and then screened for their level of resistance to G418. However, the addition of the kanamycin gene increases the size of these already large plasmids and may yield unstable vectors. Smaller vectors were constructed using a single selectable marker active both in E. coli as well as in P. pastoris. To date, two selection markers are used for this purpose: the ble gene from Streptoalloteichus hindustanus, conferring resistance to Zeocin (pPICZ series) (Higgins et ah, 1998), and the hsd gene from Aspergillus terreus, conferring resistance to Blasticidin S (pPIC6 series). In these vectors, the respective resistance genes are preceded by a S. cerevisiae TEFl gene promoter needed for transcription in P. pastoris and the synthetic EM7 promoter needed for transcription in E. coli. The genes are followed by the 3' transcription processing sequences of the S. cerevisiae CYCl gene. Direct selection of hyperresistant transformants with multicopy integration events has been claimed but not clearly proven. These vectors have several advantages; they are small and contain an extended multiple cloning site, which in most cases allows C-terminal fusion of a c-myc-tag and a His6-tag to the foreign gene. Some of these vectors have special features. The so-called E-series of vectors are designed for directional cloning by rapid Cre-based recombination (pPICZ-E and pPICZa-E). In several vectors the S. cerevisiae a-factor prepro-signal sequence has been included to allow N-terminal fusion to the foreign gene. Although these vectors were constructed for the production of secreted proteins, GPCRs fused to this sequence showed a higher production level (Schiller et ah, 2000, 2001; Weiss et al, 1995).

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TABLE IV

P. pastoris Expression Vectors for Intracellular Production or Secretion Selection marker in yeast

Properties

Ref.

Intracellular pHIL-D2 pA0815

HIS4 HIS4

pPIC3.5

HIS4

pPIC3.5K

HIS4 and kan"^

pPICZ A, B, C

ble'

pPICZ-E

ble'

pPIC6 A, B, C

bsd'

pHWOlO pGAPZ A, B, C

HIS4 ble'

AOXl-promoter AOXl-promoter; BamHl and Bglll restriction sites for in vitro multimerization of the expression cassette A0X2-promoter; multiple cloning site for insertion of the foreign gene AOXl-promoter; multiple cloning site for insertion of the foreign gene; G418selection for multicopy clones AOXl-promoter; multiple cloning site for insertion of the foreign gene; Zeocin-selection; BamHl and Bglll restriction sites for in vitro multimerization of the expression cassette; His6 and c-myc epitope tags (optional) AOXl-promoter; vector designed for in vitro recombination (echo-cloning system); zeocin-selection AOXl-promoter; multiple cloning site for insertion of the foreign gene; blasticidin-selection; BamHl and Bglll restriction sites for in vitro multimerization of the expression cassette; His6 and c-myc epitope tags (optional) GAP-promoter GAP-promoter; multiple cloning site for insertion of the foreign gene; zeocin-selection; BamHl and Bglll restriction sites for in vitro multimerization of the expression cassette; His6 and c-myc epitope tags (optional)

[1] [2]

[3] [3]

[41

[1]

[1]

[5] [1]

Secretion pHIL-Sl

HIS4

pPIC9

HIS4

AOXl-promoter; PHOl-signal sequence; multiple cloning site for insertion of the foreign gene A0X2-promoter; S. cerevisiae a-factor signal sequence; multiple cloning site for insertion of the foreign gene

[1]

[3]

69

CHAPTER 3 Production and Purification of Recombinant Membrane Proteins

TABLE IV

Continued Selection marker in yeast pPIC9K

HIS4 and kan'

pPICZaA, B, C

ble'

pPICZa-E

hie'

pPIC6a A, B, C

bsd'

pGAPZaA, B, C

ble'

Properties

Ref.

AOXl-promoter; S. cerevisiae a-factor signal sequence; multiple cloning site for insertion of the foreign gene; G418-selection for multicopy clones AOXl-promoter; S. cerevisiae a-factor signal sequence; multiple cloning site for insertion of the foreign gene; zeocin-selection; BamHl and Bglll restriction sites for in vitro multimerization of the expression cassette; His6 and c-myc epitope tags (optional) AOXl-promoter; S. cerevisiae a-factor signal sequence; vector designed for in vitro recombination (echo-cloning system); zeocin-selection AOXl-promoter; S. cerevisiae a-factor signal sequence; multiple cloning site for insertion of the foreign gene; blasticidin-selection; BamHl and Bglll restriction sites for in vitro multimerization of the expression cassette; His6 and c-myc epitope tags (optional) GAP-promoter; S. cerevisiae a-factor signal sequence; multiple cloning site for insertion of the foreign gene; zeocin-selection; BamHl and Bglll restriction sites for in vitro multimerization of the expression cassette; His6 and c-myc epitope tags (optional)

[3]

[6]

[1]

[1]

[1]

Adapted and expanded according to Higgins and Gregg (1998). [1] Invitrogen; [2] Thill et ah, 1990; [3] Scorer et ah, 1993; [4] Higgins et al, 1998; Scorer et al, 1993; [5] Waterham et al, 1997; [6] Higgins et al, 1997.

For constitutive production of foreign proteins the GAP promoter (Waterham et al., 1997) has recently been inserted in pPICZ vector backbone. The GAP promoter is a convenient alternative to the AOXl promoter as long as the expressed products are not toxic to the cell. As yet, membrane proteins have not been produced by these vectors.

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As in S. cerevisiae, linearized vectors are used for the generation of stable transformants via homologous recombination fostered by sequences shared between vector and genome. Even in the absence of an antibiotic selective pressure, such transformants are stable. The vectors mentioned above carry at least one genomic DNA fragment (the promoter) with a unique restriction site that can be cleaved and used to direct the vector to integrate into the P. pastoris genome via a single crossover. Vectors that contain the HIS4, therefore, can be forced to insert into the P. pastoris genomic his4 locus. Vectors that bear in addition the 3'AOXl sequences can be integrated in the genome by single crossover at either the AOXl or the HIS4 locus, but they can also replace the AOXl completely by a double crossover event. Recombinant clones resulting from such an event (replacement) show a His"^ Mut^ phenotype. Multiple integrations of the expression cassette occur sponataneously at low frequency (1-10% of His"^ transformants). Detection for these events is possible by screening for clones showing hyperresistance on G418 or Zeocin. As mentioned above, when the AOXl promoter is used for transcriptional control of the foreign gene, a major advantage of P. pastoris is that it can be grown to extremely high cell densities in fermenters. The AOXl promoter is induced by methanol and repressed in the presence of excess glycerol. A standard fermentation protocol is to grow the recombinant cells with an amount of glycerol, just enough to get very high cell density. When glycerol is exhausted, protein production is induced by supplementation of methanol to the medium. The advantage of Muf^ transformants is their faster growth on methanol. However, the concentration of methanol must be tightly controlled to avoid enrichment of toxic formaldehyde, the product of the first enzymatic step in the metabolism of methanol. Mut^ strains grow slowly on methanol because they lack AOXl and their growth relies on AOX2. In principal, cultivation of a Mut^ strain is similar to that of Mut^ strains, but lower methanol feed rates are needed to maintain a methanol concentration of 0.2-0.8% in the fermenter. Alternatively, Mut^ strains can be grown in a mixed glycerol/methanol medium (Stratton et al., 1998).

B. Insect Cells as Expression System Different insects cell lines for stable or transient transfection with appropriate vectors are available. Stably transformed insect cells have a number of potential benefits over mammalian cell-based expression systems (McCarroU and King, 1997; Pfeifer, 1998). These include lower growth temperature, less complex growth conditions, the possibility for a long-term continuous culture, growth in large fermenters, and the possibility of protein production in a null background. Vectors, which allow quick selection of stable cell clones, were constructed for transfection of Spodoptera frugiperda (Sf9 and Sf21 cell lines), Trichoplusia ni ("high five" cell lines), and Mamestra brassicae cell lines. Most commonly, baculovirus immediate early (ie) promoters from Bombyx mori

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multicapsid nuclear polyhedrosis virus (BmMNPV), Autographa californica (AcMNPV), and Orgyia pseudotsugata (OpMNPV) are used to drive transcription of the foreign gene (Jarvis et ah, 1990; Jarvis and Finn, 1996). Other widely used insect promoters include that of actin, of Drosophila melanogaster metallothionein, of Drosophila hsp70, and of copia, which function in the cell lines, albeit at a much lower level. The latter promoters have been mainly used for production of proteins in Drosophila, Schneider S2 and S3 cell lines, and mosquito cells. A variety of selection systems can be applied for the stable transformation of insect cells. They are based on genes conferring resistance to geneticin (G418), hygromycin B, methotrexate, and, recently, Zeocin and Blasticidin. The procedure for the generation of stably transformed insect cells is easy. Cells are seeded into small dishes and transfected with the appropriate recombinant vector using standard transfection methods such as calcium phosphate precipitation, electroporation, or liposome-mediated transfection. After a growth phase of 1-2 days to ensure expression of the resistance gene, selective pressure is applied by administration of the respective antibiotic. Nonresistant (i.e. nontransfected) cells die, and individual recombinant cell clones can be picked for further amplification. One of the most commonly used applications of stably transfected insect cells is the production of receptors or receptor subunits. Since in most cases insect cells do not appear to produce homologous endogenous receptors, the respective proteins are produced in an extremely low background. The insect/baculovirus-expression system has also proven to be successful for overproduction and characterization of several membrane proteins. The recombinant membrane proteins produced in the insect cells are subjected to posttranslational modifications such as glycosylation, phosphorylation, and palmitoylation. Most importantly, simultaneous infection of insect cells with several recombinant baculoviruses is possible. This opens the way for the production of complex multisubunit proteins or for enhancement of production levels of certain proteins by coproduction with a set of appropriate chaperons. 1. The Baculovirus Expression System The baculoviridae comprise a large family of occluded viruses, which are pathogenic for arthropods belonging to the insect orders Lepidoptera, Diptera, and Hymenoptera (Miller, 1996). In the last decade, baculoviruses, especially the Autographa californica nuclear polyhedrosis virus (AcMNPV), have been developed to a popular and versatile expression system (Kost and Condreay, 1999). The virus can be propagated easily in a multitude of cell lines, from which Spodoptera frugiperda (Sf9 and Sf21 cell lines), Trichoplusia ni ("high five" cell lines), and Mamestra brassicae are most frequently used (Hink et ah, 1991). As most baculoviruses, the AcMNPV has a complex, biphasic life cycle with

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two distinct morphological virus types (Miller, 1996). The so-called budded virus form comprises a single enveloped virus, whereas the so-called occluded virus form comprises multiple virus particles that become embedded in a protein matrix composed almost exclusively of one protein called polyhedrin. The development of the expression system was based on the dispensibility for in vitro propagation of at least two very "late" viral gene products: the plO and the polyhedrin gene. Both genes are under control of strong promoters, which were used for the design of expression vectors. However, most baculovirus expression vectors use the polyhedrin gene promoter and locus for insertion of foreign DNA. "Early" promoters have also been used for the heterologous production of proteins but were found to be less effective (Jarvis et al, 1996). The manipulation for direct cloning of the baculovirus genome, a closed, circular, double-stranded DNA molecule of about 130 kb, is a difficult task. Therefore, recombinant transfer vectors containing the flanking region of the polyhedrin gene were constructed for cloning of the foreign gene. The recombinant virus is generated by homologous recombination with these vectors. The viral DNA and the recombinant transfer vector are cotransfected, and the expression cassette recombines into the virus genome, replacing its wild-type polyhedrin gene. The efficiency of this technique is less than 1%, and the screening for recombinant virus, which is performed by plaque assays a n d / o r hybridizations, is tedious and time-consuming (Luckow and Summers, 1988). By using linearized virus DNA for cotransfection, the efficiency was raised to about 30% recombinants per virus progeny. For this approach a unique Bsw36I restriction site was introduced at the polyhedrin locus of wild-type baculovirus DNA (Kitts et ah, 1990). A further development was the use of the baculovirus designated BacPAK6 for cotransfection. This virus DNA contains the E. coli lacZ gene at the polyhedrin locus and two additional Bsu36l sites in the polyhedrin flanking regions (Kitts and Possee, 1993). After Bsu36l digestion the virus DNA is not only linearized but two genomic fragments are removed. One disrupts a gene essential for replication of the virus. Restoration of this gene is only possible after recombination of the linearized and substracted virus genome with the respective polyhedrin-flanking region in the recombinant cotransfected transfer vector. The selection of recombinant baculovirus is based on LacZ (blue/white assay). Recombinant baculoviruses form white plaques on a background of blue plaques. Due to these modifications the efficiency of the system was raised to 80-90% recombinants per virus progeny. The system is commercially available (BaculoGold, Pharmingen). An alternative system is based on linearized BacPAK6 DNA (Orbigen). To increase expression, the gene encoding for protein disulfide isomerase has been inserted at the plO locus of the virus (Sapphire Baculovirus DNA). This might be advantageous for the production of membrane proteins. Another popular system is based on a site-specific transposition of an expression cassette into a baculovirus, which propagates in E. coli (Luckow et

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al, 1993, Invitrogen). Here, the complete baculovirus genome replicates as a large and stable, low-copy number bacmid (pMON14272) in E. coli. This was achieved by recombination of a mini-F replicon into the polyhedrin locus of the virus. Additionally, the target site for the Tn7 transposon, a kanamycin resistance gene, and the lacZoc peptide coding region originating from a pUC-based vector have been introduced into the bacmid. The attachment site for the Tn7 transposon (mini-affTn7) was inserted corresponding to the N-terminus of the lacZoc region without disrupting the reading frame. Transposition of an expression cassette is performed in the E. coli DHlOBac strain harbouring the bacmid as well as the tetracyclin-resistant helper plasmid pMON7124, which in-trans supplies proteins necessary for transposition. The cells are transformed with an ampicillin-resistant vector that contains the foreign gene cloned under the transcriptional control of the polyhedrin promoter and the gentamycin acetyltransferase gene flanked by the left and right arms of Tn7. The mini-Tn7 in the so-called pFastBac vectors, which also contain the inserted foreign gene, can transposit to the vmni-attTn? attachment site on the bacmid. This transposition disrupts the lacZa peptides so that colonies, which contain a recombinant bacmid, are white in a background of blue colonies. Recombinant transformants are selected for in the presence of kanamycin, tetracyclin, gentamycin, X-Gal, and IPTG. The bacmids are isolated from the transformants and directly used for transfection of insect cells, eliminating the need for a tedious screening of positive plaques. In addition, the time required to generate, identify, and characterize a recombinant baculovirus with the transposition-based baculovirus system is reduced to about a week as opposed to six weeks with the former approach. For the procedures introduced here, different vectors are available, some of which also allow fusions to signal sequences, different tags (His-tag, c-myc-tag), as well as other proteins (GST). Some vectors allow simultaneous production of two or three proteins from one expression cassette via multiple promoters and multiple cloning sites.

IV. USE OF FUSION PROTEINS AND AFFINITY TAGS FOR PURIFICATION OF MEMBRANE PROTEINS Peptide affinity tags and fusion proteins are valuable tools that allow efficient and standardized purification of recombinant proteins. These affinity markers are attached to the target protein by gene fusion, thereby adding specific properties to the recombinant protein that can be exploited for protein purification via affinity chromatography, as well as for easy detection and immobilization. Affinity chromatography systems are designed to be highly specific and fast. They allow protein purification with high-yield, highenrichment, and often mild-purification conditions. In general, affinity tags

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and fusion proteins can be added to the amino(N)-terminus or the carboxyl(C)-terminus of the protein. Alternatively, the DNA encoding the respective affinity marker may be introduced within the coding sequence of the target protein, a so-called internal fusion. The latter approach requires information about the secondary structural elements and the topology of the target. In some cases, linker regions have to be included to ensure accessibility of the affinity marker. The introduction of a tag or a fusion protein should affect neither the activity or structure of the protein of interest nor interfere with targeting, folding, or membrane insertion of the protein. In some cases, destabilization of the product and low yield are provoked, since the marker may disturb folding processes of the protein or increase its susceptibility to proteolytic degradation. The prediction of disadvantageous side effects is difficult, and, in practice, each affinity system has to be established empirically. Therefore, it is recommended to prepare different gene arrangements with respect to type and location of the tag and the length of the linker region. Short affinity tags are less likely to interfere with structure and function of the target protein and might be preferred for this reason. However, a stabilizing effect of larger fusion proteins has been reported and might be desirable. The insertion of a highly specific protease cleavage site between protein and affinity tag or fusion domain allows the removal of the attached marker after protein purification. The most common examples of affinity tags and fusion proteins used for membrane proteins will be described in this section. A general requirement for this application is tolerance to detergents. The Hzs-tag is probably the most widely applied affinity tag. Six or 10 consecutive histidine residues are fused to either the N- or the C- terminus of the protein. The tagged protein can be purified by immobilized metal-affinity chromatography (IMAC, Hochuli et ah, 1988). For this purpose metal-chelating matrices, which are loaded with transition metal ions (Co^"^, Ni^"^, Cu^^, Zn^), are used. The hybrid protein binds by interaction of the histidine imidazole groups with the free ligand binding sites in the coordination sphere of the metal ions. After applying the protein mixture to the column, unbound material is washed off with an appropriate buffer. The specifically bound protein is eluted by adding free imidazole to the elution buffer or by lowering the pH. This rapid method allows 100-fold enrichments in a single purification step and results in u p to 95% purity of the affinity-tagged protein (for review, see Bornhorst and Falke, 2000). It has been applied for purification of recombinant proteins from a number of different expression systems. The most commonly used H/s-tag consists of six histidine residues. Longer histidine stretches allow more stringent washing conditions and might help to reduce impurities. However, the longer the tag, the higher the risk that it interferes with the protein function. Affinity matrices are commercially available—for example, Nickel-nitrilotriacetic acid (Ni^"^-NTA, Qiagen) or cobaltcarboxymethylaspartate (Co^'^-CMA or Talon, Clontech). The binding affinity

CHAPTER 3 Production and Purification of Recombinant Membrane Proteins

75

is very high compared to other affinity systems. IMAC materials are reusable, since the metal ions can be stripped off and reloaded for regeneration. A binding capacity of standard affinity matrices of 5-10 mg of protein per ml of matrix is described. Furthermore, the matrices tolerate the presence of detergents and can be used easily for the purification of membrane proteins as, for example, the sodium/proton antiporter from E. coli (Olami et ah, 1997), the plant plasma membrane H"^-ATPase (Jahn et ah, 2001), and the trehalose/ maltose ABC transporter (Greller et ah, 2001). Remarkably, the siderophore receptor FhuA located in the outer membrane of E. coli was successfully purified with an internal H/s-tag. A hexahistidine peptide was inserted in a loop that was known to be surface exposed. This approach allowed purification of the receptor to homogeneity in large quantities and consequently its crystallization suitable for x-ray analysis (Ferguson et ah, 1998). Rogl et ah (1998) succeeded in refolding the chloroplast light-harvesting complex II from inclusion bodies, while the protein was immobilized on an IMAC matrix. In addition, the His-tag is often used in multiaffinity fusion systems, in which several tags or fusion proteins are attached to the same protein (Bornhorst and Falke, 2000). The Strep-tag is another useful but, until today, less well-known affinity marker. The short peptide consists of nine amino-acids and binds to streptavidin, allowing one-step purification of the fusion protein via streptavidin affinity chromatography (for review, see Skerra and Schmidt, 2000). The Sfrep-tag binds to the biotin binding pocket of streptavidin and permits very gentle elution of the recombinant protein from the affinity matrix by competitive displacement. Already small amounts of biotin or analogues thereof are sufficient for efficient elution without changing the overall buffer conditions with respect to pH or ionic strength. This second specificity-conferring step enables purity grades of over 99% in one step. While the original Sfrep-tag with the sequence Ala-Trp-Arg-His-Pro-GlnPhe-Gly-Gly (Schmidt and Skerra, 1993) can only be used as C-terminal fusion, the slightly modified version Strep-tag II (Asn-Trp-Ser-His-Pro-GlnPhe-Glu-Lys) allows in addition N-terminal and internal addition of the affinity tag. Affinity matrices can either be custom-made by coupling commercially available streptavidin to a variety of matrices or they can be obtained ready-made. Strep-Tactin columns (IBA) consist of an engineered streptavidin mutant with optimized binding properties for the Strep-tag II (Voss and Skerra, 1997) immobilized to various matrices, enabling affinity purification via gravity flow, FPLC, or HPLC. Binding of Strep-tagged proteins to streptavidin matrices is highly specific. Thus, it is an attractive alternative to Hfs-tagged proteins when problems with unspecific contamination cannot be solved. Specific elution of the protein from streptavidin matrices is most often performed with desthiobiotin. Biotin itself binds irreversibly to tetrameric streptavidin and thus does not allow the reversible use of the affinity matrix. To avoid contamination with endogenous biotin or biotin-carrying proteins.

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extracts can be pretreated with avidin that will irreversibly bind and thereby mask biotin, while the Strep-ieig is not recognized. The Strep-tag system has been used for the purification of a variety of recombinant proteins, although applications described up to now comprise mainly proteins from bacterial sources. It seems to be a promising approach for membrane protein purification, since the streptavidin affinity matrix tolerates various detergents, the binding properties are highly specific, and the elution conditions are very mild. The betaine carrier BetP from Corynebacterium glutamicum was successfully purified by streptavidin affinity chromatography (Riibenhagen et ah, 2000). Furthermore, the Strep-tag has already been proven to be suitable for the purification of multisubunit membrane protein complexes, as, for example, the cocomplex of cytochrome c oxidase from Paracoccus denitrificans with the specifically bound antibody fragment carrying the Strep-tag (Ostermeier et ah, 1995). 3D crystals of this protein preparation (Ostermeier et ah, 1995) led to the structure determination of the P. denitrificans cytochrome c oxidase (Iwata et ah, 1995). The Flag-tag is an epitope tag that can be used for membrane protein purification. This peptide consists of eight amino acid residues (Asp-Tyr-LysAsp-Asp-Asp-Asp-Lys) and forms the epitope for a set of different monoclonal antibodies. The monoclonal antibody Ml reacts specifically with the Flag-tag at the free N-terminus of the protein, and the binding is calcium dependent. Therefore, Flag-tagged proteins can be bound to a mAB Ml affinity matrix in the presence of calcium and eluted by adding EDTA to the buffer. Furthermore, the N-terminal specificity offers an elegant method to select for recombinant proteins with correctly processed leader sequences (see Chapter 9) or for selection of homogenous products after cleavage of Nterminal fusions. The mAB M2 binds to the Flag-epitope at the N- or C-terminus of the protein, but the binding is not calcium dependent. Elution of protein bound to mAB M2 affinity columns can be achieved by pH shift or by competition with the free Flag-peptide. Antibodies as well as specific affinity matrices are commercially available (Eastman Kodak). In vivo biotinylation can be exploited for protein purification. Biotinylation occurs in all eukaryotic and prokaryotic organisms, and the number of different endogenous biotinylated proteins is low. The protein of interest is fused to a Bio-tag that provides the biotin (vitamin H) accepting domain either in peptide or protein domain format. The highly specific interaction of biotin with streptavidin or avidin allows selective purification of the marked protein. The system can be used in principal for all expression systems and different biotin acceptor domains have been successfully introduced. Biotinylated proteins bind to monomeric avidin with a dissociation constant of about 10"^ M compared to 10"^^ for the tetrameric form. Therefore, immobilized monomeric avidin is often used as affinity matrix, and elution can be easily accomplished by competitive elution with low concentrations of biotin (for review, see Cull and Schatz, 2000). This system was used, for example, for the

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purification of the jS2-adrenergic receptor, which was fused to a 75 amino acid biotinylation domain of Propionibacteriutn shertnanii and expressed in Pichia pastoris (see Chapter 9). The rat neurotensin receptor expressed in E. coli was purified most efficiently with a 13-amino-acid-long Bzo-tag at its C-terminus (Tucker and Grisshammer, 1996). Various fusion proteins — for example, the maltose-binding protein (MBP), the jS-galactosidase (GAL), thioredoxin (TRX), glutathione S-transferase (GST), or protein A—have been attached to proteins to facilitate purification. The fusion of larger domains offers the advantage of not only providing an affinity marker but it often stabilizes the recombinant protein by preventing degradation or promoting solubility. The N-terminal fusion of thioredoxin was essential for stabilizing the membrane-bound protein cytochrome b (5), whereas the C-terminal Hzs-tag was used for purification (Begum et ah, 2000). Again, a stabilizing effect of thioredoxin was reported for the production of the rat neurotensin receptor expressed in E. coli (Tucker and Grisshammer, 1996). In addition, thioredoxin can be directly applied for purification by either using its inherent thermal stability or by introducing secondary affinity properties to thioredoxin—for example, with in vivo biotinylation (for review, see LaVaUie et ah, 2000). Hybrid proteins containing the MBP fusion can be easily purified by affinity chromatography using immobilized amylose matrix (for review, see Sachdev and Chirgwin, 2000). This method has been successful for the purification of membrane proteins (Hampe et ah, 2000; Henderson et ah, 2000). GST fusion proteins are commonly used for protein purification from E. coli sources. The corresponding affinity matrix is glutathione-agarose. Bound protein can be eluted by adding glutathione to the buffer. No standard protocols can be applied, but conditions for purification have to be carefully optimized (for review, see Smith, 2000). A truncated form of the human Na"^/glucose cotransporter was expressed and purified from E. coli using a GST fusion vector and glutathione affinity chromatography (PanayotovaHeiermann et ah, 1999). When constructing the expression cassette for the hybrid protein, it is advisable to include a short sequence for a specific protease site between the target protein and the affinity fusion protein. Specific proteases such as thrombin, factor Xa, or Tobaco Etch Virus (TEV) protease (Dougherty et al., 1989) are used for the selective removal of the fusion partner, a step that might be exploited for purification.

ACKNOWLEDGMENTS Thanks are due to Danieli Tsafi (Protein Expression Facility), Institute of Life Sciences, Hebrew University of Jerusalem, Givat Ram for help and advice, and to the support by the BMBF and International Bureau of the BMBF at the

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DLR (German-Israeli Project Cooperation of Future-oriented Topics) and the Israel Science Foundation. Financial support by the Deutsche Forschungsgemeinschaft (SFB 472) to C.H. is acknowledged.

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