Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci

Efficient enrichment of staphylococcal cells displaying specificheterologous affinity ligands on their cell surfaces was demonstratedby using fluorescence-activated cell sorting. Using bacterialsurface display of peptide or protein libraries for the purposeof combinatorial protein engineering has previously been investigatedby using gram-negative bacteria. Here, the potential for usinga gram-positive bacterium was evaluated by employing the well-establishedsurface expression system for Staphylococcus carnosus. Staphylococcusaureus protein A domains with binding specificity to immunoglobulinG or engineered specificity for the G protein of human respiratorysyncytial virus were expressed as surface display on S. carnosuscells. The surface accessibility and retained binding specificityof expressed proteins were demonstrated in whole-cell enzymeand flow cytometry assays. Also, affibody-expressing targetcells could be sorted essentially quantitatively from a moderateexcess of background cells in a single step by using a high-stringencysorting mode. Furthermore, in a simulated library selectionexperiment, a more-than-25,000-fold enrichment of target cellscould be achieved through only two rounds of cell sorting andregrowth. The results obtained indicate that staphylococcalsurface display of affibody libraries combined with fluoresence-activatedcell sorting might indeed constitute an attractive alternativeto existing technology platforms for affinity-based selections.

INTRODUCTION

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Introduction

Recent advances within the field of combinatorial protein engineeringhave led to the development of several complementary technologiesfor the selection of novel protein variants from large libraries.So far, phage display has been the preferred format for directedevolution efforts (39), but more recently, other techniques,such as ribosomal display (29), covalent display, and differentformats of cell display, have become attractive alternatives(46). Cell surface display combined with fluorescence-activatedcell sorting (FACS) constitutes a powerful strategy for isolationof novel ligands with improved affinity, stability, or enzymaticactivity (4, 28, 37). The high throughput and quantitative multiparameterpopulation analysis of modern flow cytometers makes FACS idealfor protein engineering applications (7, 46). However, FACSsorting would only be applicable with cell display systems,since the phage particles are too small to be sorted with thepresent state-of-the-art flow cytometers (6, 43).

Traditionally, engineering of antibody fragments has been thedominating strategy for generating novel proteins with specificligand-binding properties (17). More recently, other proteinscaffolds have also been investigated as sources for novel affinityligands (26, 38). One such novel class of affinity proteins,called affibody ligands, i.e., engineered Staphylococcus aureusprotein A (SpA) domains, has recently been described (23). Combinatoriallibraries were created through simultaneous randomization of13 amino acid residues. Through the genetic fusion of theselibraries to the coat protein III of filamentous phage M13,phage libraries adapted for the selection of novel affinityvariants were created. This strategy has been successfully usedto select affibody ligands to diverse targets, including TaqDNA polymerase, human recombinant factor VIII, a human apolipoproteinvariant, and the G protein of human respiratory syncytial virus(RSV) (15, 23, 25). The same strategy has also been employedto select affibody ligands to immunoglobulins (Igs) for whichSpA has no inherent affinity, e.g., human IgA and IgE (12, 30).So far, phage display has been used for selection of affibodyligands, but a number of bacterial expression systems wouldalso be available (3, 18, 33). Regarding bacterial display forprotein engineering purposes, combinatorial libraries have sofar been displayed only on gram-negative bacteria but inherentmorphological properties of gram-positive bacteria would makethem an attractive alternative in this context (40). These propertiesinclude the observed high viability and robustness of staphylococcalcells in high-speed flow cytometry cell sorting (40, 42) andthe convenience of C-terminal anchoring of proteins on the cellsurface that is characteristic of gram-positive bacteria (33),which allows insertion of extended sequences without interferencewith the translocation machinery.

Two previously described systems for cell surface display onthe nonpathogenic food-grade bacteria Staphylococcus carnosus(34) and Staphylococcus xylosus (16) have been extensively investigatedin various biotechnological applications (43), most frequentlyfor development of live bacterial vaccine delivery vehiclesthrough the display of heterologous immunogens at the cell surface(6, 40). Furthermore, single-chain Fv antibody fragments, aswell as IgE- and IgA-specific affibody ligands, have been displayed,creating potential whole-cell diagnostic devices (12, 13). Inaddition, staphylococci with increased metal-binding capacityhave been created through display of polyhistidyl peptides (35),and novel metal-binding proteins have been generated throughcombinatorial protein engineering approaches (19, 44).

An improved S. carnosus display vector exhibiting enhanced growthcharacteristics and DNA stability was recently developed (45)for intended use in future display of peptide and protein librariesand subsequent affinity-based selections of fluorescence-labeledtarget cells by flow cytometry. The observed high viabilityand robustness of the staphylococcal cells in the flow cytometermight be of considerable importance for this type of application(45). Here, we report the use of a model system employing surface-displayedengineered SpA domains to test the functionality of this S.carnosus display system for the specific enrichment of targetcells from a large excess of background cells by flow cytometry.The accessibility and binding specificity of affibody ligandsexposed on the surface were analyzed in whole-cell enzyme assays,and target cells were enriched by using single or multiple roundsof sorting and reamplification by growth. Implications for thedisplay of whole affibody libraries and subsequent affinity-basedselections of novel binding proteins will be discussed.

MATERIALS AND METHODS

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Materials and Methods

Plasmid constructions.
The Escherichia coli strain RRI ${Delta}$ M15 (31) was used as the hoststrain during plasmid constructions. Gene fragments correspondingto an engineered IgG-binding SpA domain, Z_wt (22), or to anSpA-based affibody binding to the G protein of human respiratorysyncytial virus (RSV), Z_RSV1 (15), were amplified by PCR. The5' PCR primer SAPA27 introduced a BamHI restriction enzyme sitein the upstream region of the affibody sequence. Similarly,the 3' primer SAPA28 added a SalI restriction enzyme site inthe downstream region. The generated PCR fragments were ligatedinto the surface display vector pSCXm, which had previouslybeen digested with BamHI and SalI (45), thus generating theplasmids pSCXZ_wt and pSCXZ_RSV1, respectively. Positive cloneswere identified by PCR screening and verified by DNA sequencingperformed on the Mega-BACE 1000 DNA sequencing system (AmershamBiosciences, Uppsala, Sweden) by using MegaBACE terminator chemistry(Amersham Biosciences) in a cycle-sequencing protocol.

Preparation of biotinylated human IgG.
Human IgG was biotinylated by using the EZ-Link Sulfo-NHS-LC-Biotinlabeling kit (Pierce, Rockford, Ill.) according to the supplier'srecommendations. The unreacted free biotin was removed by Microconcentrifugal filter devices (Millipore Corp., Bedford, Mass.).The final concentration of IgG-biotin was determined by a standardBradford analysis using bovine serum albumin as a standard.

Evaluation of surface display efficiency and IgG binding.
Overnight cultures of recombinant and wild-type staphylococciwere diluted 1:200 in growth medium (containing chloramphenicolwhen appropriate) and grown at 37°C to an A₅₇₈ of 1. Thecells were harvested and washed twice with phosphate-bufferedsaline (PBS) supplemented with 0.05% Tween 20 (PBST) (pH 7.5)before being resuspended in PBST to an A₅₇₈ of 1. One-milliliteraliquots from these suspensions were incubated at room temperaturewith either biotinylated human serum albumin (HSA) (1.75 µlof biotinylated HSA [2.39 mg/ml] in 1 ml of PBST) or biotinylatedIgG (4 µl of biotinylated IgG [11.5 µM] in 1 mlof PBST) for surface expression and binding analysis. The cellswere washed twice in PBST prior to resuspension in 1 ml of PBSTcontaining 0.5 units of streptavidin-alkaline phosphatase conjugate(Boehringer, Mannheim, Germany) and then incubated for another30 min at room temperature. After two additional washings, thecells were diluted 1:10 in substrate buffer (1 M diethanolamine-HCl[pH 9.8], 0.5 mM MgCl₂), and five 100-µl aliquots of eachcell type were loaded into a microtiter plate before additionof 100 µl of the substrate solution (p-nitrophenylphosphate;Sigma, St. Louis, Mo.). The change in A_{405 nm} was measured for20 min in an enzyme-linked immunosorbent assay plate reader(SUNRISE, Tecan, Grödingen, Austria).

FACS.
Overnight cultures grown at 37°C were harvested and washedtwice in PBS before being resuspended at an optical densityat 600 nm of 1 in PBS. Positive (pSCXZ_wt) and negative (pSCXZ_RSV1)cell populations were mixed at the desired ratios, and 100-µlaliquots were withdrawn, pelleted, and resuspended in a solutionof 14 nM biotin-IgG in PBS. After incubation for 30 min on ice,the cells were washed once with ice-cold PBS before being resuspendedin Alexa-Fluor⁴⁸⁸-streptavidin (Molecular Probes, Eugene, Oreg.)conjugate diluted 1,600x in PBS. Following another incubationfor 30 min on ice, the cells were washed and resuspended inPBS at approximately 10⁷ cells per ml and either counted orsorted on the basis of fluorescence intensity with a FACS VantageSE flow cytometer (Becton Dickinson, Sunnyvale, Calif.). Thecytometer was set to enhanced normal-R mode for the single-stepenrichments and to enrich for the library simulations. The cellswere sorted directly into tryptic soy broth medium and eitherplated onto chloramphenicol plates or reamplified by incubationovernight and subjected to another round of cell sorting andanalysis. After incubation at 37°C for 48 h, the sortedcells were screened by PCR by using primer pairs specific forthe genes encoding the Z_wt and Z_RSV1 variants. The fractionof sorted cells expressing the desired variant could thus bedetermined.

RESULTS

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Results

Background.
In this study, we have explored the possibility of using staphylococcalsurface display and flow cytometry as an alternative to filamentousphages for affinity-based selection of engineered SpA domains,the so-called affibody ligands. A previous study demonstratedthat affibody ligands were efficiently displayed with retainedbinding specificity on the surfaces of S. carnosus cells (12).Recently, the plasmid vector for surface display on S. carnosuswas significantly improved with respect to genetic stabilitywith retained surface expression efficiency (45) in order tobe better suited for library selection applications. This increasedstability should potentially enable the display of whole affibodyligand libraries at the cell surface. In this study, a modelsystem consisting of the IgG-binding affibody scaffold, Z_wt(22, 24), and a previously described affibody, Z_RSV1 (15), selectedusing phage display and specific for the G protein of RSV, wasused for simulated library selections.

Display vectors.
Two novel plasmid vectors were constructed for display of chimericproteins containing the Z_wt or Z_RSV1 affibody ligands (15, 24)on the S. carnosus cell surface. In Figure 1, the parental vector,pSCXm (45), as well as the two novel constructs, pSCXZ_wt andpSCXZ_RSV1, are schematically depicted together with their encodedgene products as anchored to the staphylococcal cell wall. Forsimplicity, the recombinant S. carnosus strains were designatedSc:m, Sc:Z_wt and Sc:Z_RSV1. The pSCXm display vector utilizesthe promoter, signal sequence, and propeptide from a Staphyloccocushyicus lipase for optimized expression and secretion in S. carnosus(20). The lipase propeptide, which is cleaved off in its homologoushost, S. hyicus (2), but not in S. carnosus (9), has been extensivelystudied and proved to be essential for efficient secretion ofheterologous gene fusion products in the lipase expression system(8, 32). The vector system also contains the gene fragmentsX and M from the C-terminal cell wall anchoring part of theSpA gene for covalent anchoring of expressed proteins to thepeptidoglycan cell wall (21, 36, 42). In addition, the geneencoding an albumin binding protein (ABP) (27), derived fromstreptococcal protein G, is also present in the expression vector.The ABP region is expressed as the part of the chimeric surfaceprotein closest to the cell wall-anchoring motifs functioningas a spacer molecule, increasing the accessibility of expressedchimeric proteins at the bacterial surface (41). Also, ABP-containingsurface proteins can be extracted from the cell wall by lysostaphintreatment and can subsequently be purified by affinity chromatographyon HSA resins (31), utilizing the ABP moiety as an affinityhandle. Using this method is a convenient strategy for verifyingthat the chimeric surface proteins are expressed as full-lengthproteins and not subjected to degradation. Finally, the ABPtag functions as a reporter molecule in enzyme- and flow cytometry-basedwhole-cell assays (34, 45) for monitoring expression levelsand density of surface-displayed receptors.

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FIG. 1. The expression cassettes of the different expression vectors suitable for surface display on S. carnosus together with a schematic picture of the processed gene fusion products, illustrated as anchored to the cell surface. The names of the constructed expression vectors are given below the expression cassettes, and the abbreviated names of the recombinant staphylococci are shown to the right. When a target gene is introduced into the multiple cloning sites of the vector, the encoded target protein is exposed, as illustrated, anchored to the staphylococcal cell surface. M' represents the processed and covalently anchored form of the M sequence of SpA. Note that the propeptide (PP) from S. hyicus is not processed in S. carnosus. Abbreviations: S, secretion signal from S. hyicus lipase; Bla, gene encoding ß-lactamase; Cml, gene encoding chloramphenicol acetyltransferase; OriE, origin of replication from Escherichia coli; OriS, origin of replication from S. aureus; P_Lip, S. hyicus promoter region designed for S. hyicus lipase production in S. carnosus; X, charged repetitive region postulated to interact with the peptidoglycan cell wall.

Surface expression and IgG-binding analysis.
Before fluorescence-activated cell sorting could be performed,it was necessary to determine whether the Z_wt and Z_RSV1 domainswere efficiently expressed in a functional form at the cellsurface. In order to analyze the expression levels and accessibilityof the displayed affibody ligands, the ABP region was used asa reporter molecule in a previously described whole-cell enzymeassay (34). Briefly, recombinant and wild-type staphylococcalcells were grown to early logarithmic phase and incubated withbiotinylated HSA, followed by incubation with a streptavidin-alkaline-phosphataseconjugate. The presence of surface proteins containing the ABPdomain could then be detected through the addition of a chromogenicsubstrate. The results, shown in Fig. 2A, indicate that cellsharboring the pSCXZ_wt or pSCXZ_RSV1 plasmid express ABP-containingsurface proteins at a level similar to that of cells containingthe parental pSCXm plasmid. In analogy with previous studies,this would mean that there are approximately 10,000 ABP-containingproteins displayed per cell, enabling the use of flow cytometryfor sorting for affinity-based selection (1). The wild-typeS. carnosus cells, not expressing any albumin-binding domains,were, as expected, negative in this assay (Fig. 2A). The IgG-bindingcapacity of the recombinant strains was analyzed in a similarassay by replacing the biotinylated HSA with biotinylated IgGin the probing step. The Sc:Z_wt cells were positive in the assay,while Sc:wt, Sc:m, and Sc:Z_RSV1 cells demonstrated only backgroundreactivity (Fig. 2B), which shows that the Z_wt domain may beefficiently expressed in a functional form on the cell surfacewith retained binding specificity. The results also demonstratedvery low levels of unspecific interactions with wild-type S.carnosus cells and recombinant strains expressing non-IgG-bindingaffibody ligands.

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FIG. 2. Histogram representation of the results of colorimetric assays for determining the accessibility and IgG-binding capacity of affibodies displayed on cell surfaces. (A) Results for detection of the accessibility of receptors displayed on the cell surface and containing regions with albumin-binding capacity. Wild-type and recombinant S. carnosus cells were incubated with biotinylated HSA for binding to successfully exposed receptors containing ABP. Streptavidin-alkaline phosphatase conjugate was added, and absorbance at 405 nm was monitored after addition of substrate. (B) Results for detection of IgG-binding capacity by using biotinylated IgG in an assay similar to that used for panel A. Wild-type cells (Sc:wt) were used together with pSCXm-transformed S. carnosus cells (Sc:m) as controls in all experiments. OD, optical density.

Single-step FACS enrichment of Z_wt-expressing cells.
Whether working with the screening of combinatorial librariesor classical protein purification applications, making a calculatedchoice between yield and purity is necessary. For library screeningapplications, the goal is to obtain the purest possible productwithout losing any valuable target cells. Thus, when workingwith library selections, the maximum attainable single-stepenrichment factor becomes an important parameter. In an initialstudy, we therefore investigated the possibility of quantitativesorting of Sc:Z_wt cells from a moderate excess of control cells.Cells expressing a previously selected affibody ligand, Z_RSV1,capable of specific recognition of the G protein of RSV servedas a relevant nontarget background population. Specifically,overnight cell cultures of Sc:Z_wt target cells were mixed withSc:Z_RSV1 control cells at ratios of 1:1, 1:100 and 1:1,000.The cell mixtures were incubated with biotinylated IgG and anAlexa-Fluor⁴⁸⁸-streptavidin conjugate before being analyzedand sorted on the basis of fluorescence. Since these initialsortings aimed at obtaining a high degree of purity in a singlesorting round, a relatively narrow sorting gate was set to minimizethe number of nontarget background cells being sorted. The fractionof the total cell population that was sorted corresponded toapproximately 50, 1, and 0.1%, respectively, for the 1:1, 1:100,and 1:1,000 mixtures (data not shown). The cells were collectedand filtered through a sterile filter and applied to chloramphenicolplates. To verify that the sorted cells were indeed Sc:Z_wt,PCR primer pairs specific for either Z_wt or Z_RSV1 were usedfor clone-specific PCR amplification and screening purposesdirectly on staphylococcal colonies. The results, presentedin Table 1, clearly indicate that essentially quantitative sortingof target cells may be obtained from a moderate excess of backgroundcells in a single sorting round. For the 1:1 mixture, 100% ofthe analyzed cells were identified as Sc:Z_wt, while the numbersfor the 1:100 and 1:1,000 mixtures were 95 and 80%, respectively.This means there was a 571-fold enrichment of Z_wt-expressingtarget cells in a single sorting round for the 1:1,000 mixture.Furthermore, the high viability observed for the sorted staphylococcalcells suggests a possible advantage of using gram-positive bacteriafor cell-sorting applications. More than 95% of the analyzedand sorted cells were capable of growth on chloramphenicol plates(Table 1), indicating that the inherent structure of the staphylococcalcell wall, i.e., the thick peptidoglycan layer, indeed seemsto improve ability to withstand the elevated shear forces actingon the individual cells in the flow cytometer.

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TABLE 1. Single-step enrichment of Z_wt-expressing staphylococci at different Z_wt:Z_RSV1 ratios

Sorting of very rare target cells from a large background.
The possibility of also using FACS for the isolation of veryrare target cells from a large excess of background cells wasevaluated. S. carnosus cells expressing surface displays ofZ_wt or Z_RSV1 affibody ligands were mixed at a ratio of 1:100,000and labeled with biotinylated IgG and Alexa-Fluor⁴⁸⁸-streptavidinconjugate to create a simulated library situation. In this experiment,the starting mixture was incubated with the fluorescence-labeledprobes, sorted on the basis of fluorescence, and enriched throughmultiple rounds of cell sorting and amplification by overnightgrowth (Fig. 3). Since the initial number of positive cellsin this experiment was very small, a wide sorting gate was setso as not to lose any valuable target cells. Initially, approximately0.2% of the control Sc:Z_RSV cells fell within the sorting window(Fig. 4C), thus ensuring that most of the target cells wereeffectively sorted in the first sorting round. One million cellsfrom the input mixture were run through the flow cytometer,and Z_wt-expressing target cells that fell within the sortingwindow were collected and grown overnight in fresh medium andre-sorted. The cell fluorescence distribution after two roundsof sorting and regrowth are shown in Fig. 4C through E, andthat of the positive and negative control populations is shownin Fig. 4A and B. The bar in each graph represents the sortinggate setting, i.e., the minimum fluorescence intensity definedas a positive event, with the results for nonstained Sc:Z_RSV1bacterial cells displayed to the left in the histograms andthe results for cells exposing Z_wt-containing proteins on theirsurfaces shifted to the right. Approximately 26% of the cellsfell within the positive window (Fig. 4E), which correspondsto a more-than-25,000-fold enrichment of target cells in onlytwo rounds of cell sorting. The identities of the sorted cellswere further analyzed by PCR screening as before, confirmingthe identity of selected cells (data not shown). These datademonstrate that specific enrichment of rare target cells canbe efficiently achieved by FACS sorting by using the above staphylococcaldisplay system. Importantly, the observed enrichment is notdue to a growth advantage of the Z_wt-expressing S. carnosuscells, since repeated regrowth of the starting mixture withoutin-between cell sorting did not result in any significant biaswhen analyzed by PCR screening using Z_wt-specific primers (datanot shown).

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FIG. 3. General procedure for the enrichment of fluorescence-labeled, Z_wt-expressing target cells by flow cytometry. Staphylococcal cells with surface display of affibodies are mixed at the desired ratios and incubated with fluorescence-labeled probes. Target cells are selected on the basis of fluorescence and enriched through multiple rounds of cell sorting and amplification by growth. The cells are plated, and individual clones are screened by PCR for identification.

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FIG. 4. Histograms of results from cell-sorting experiments. Wild-type and recombinant S. carnosus cells were probed with biotinylated IgG and stained with an Alexa-Fluor⁴⁸⁸-streptavidin conjugate. The bar in each graph represents the sorting gate setting, i.e., the minimum fluorescence intensity defined as a positive event. (A) Sc:Z_RSV1 sample used as a negative control; (B) Sc:Z_wt sample used as a positive control; (C) a 1:100,000 mixture of Sc:Z_wt to Sc:Z_RSV1 cells analyzed prior to the first cell-sorting round; (D) a 1:100,000 mixture of Sc:Z_wt to Sc:Z_RSV1 cells recovered from the first cell-sorting round; (E) a 1:100,000 mixture of Sc:Z_wt to Sc:Z_RSV1 cells recovered from the second cell-sorting round. The histograms show results for nonstained Sc:Z_RSV1 bacterial cells to the left and results for cells exposing Z_wt-containing proteins on their surfaces shifted to the right. Fluorescence intensity is shown on the x axis, and the number of cells is shown on the y axis.

DISCUSSION

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Discussion

In this study, we have demonstrated the possibility of usingstaphylococcal surface display and flow cytometry as a potentialalternative to filamentous phages for the specific and quantitativeisolation of novel affinity pairs from surface-displayed affibodylibraries. Recombinant S. carnosus strains with engineered SpAdomains specific for IgG or the G protein of human RSV exposedon the surface were created. The chimeric surface proteins wereshown to be expressed in an accessible form with retained bindingspecificity at the staphylococcal surface by an enzyme assaybased on the ABP domain. Also, affibody-expressing target cellscould be sorted essentially quantitatively from a moderate excessof nontarget staphylococci in a single sorting round by usinga high-stringency sorting mode. Furthermore, by using a simulatedlibrary situation, a more-than-25,000-fold enrichment of targetcells was achieved through only two successive rounds of cellsorting and regrowth. This indicates that real affinity-basedselections from surface-displayed affibody libraries would befeasible. In addition, the staphylococcal cells seem to be highlysuited for flow cytometry applications, since they also demonstratea high degree of viability after high-speed sorting. More than95% of the sorted cells grow well on chloramphenicol platesafter sorting. This capability is probably due to their highresistance to shear forces related to the thick peptidoglycancell wall.

One important advantage of cell-based display systems comparedto monovalent phage display is the apparent lack of avidityeffects. While phage display systems based on pIII fusions relyon single molecular binding events, a so-called monovalent display,the staphylococcal display system described here typically expressesaround 10,000 copies of the displayed protein per cell. Thismeans that random variation in expression levels or stabilityshould not interfere with library selections. Furthermore, thepresence of the ABP reporter molecule in the surface chimeraswould make it possible to normalize ligand binding by takinginto account the number of surface proteins per cell, avoidingartifacts related to expression biases when working with smalldifferences in affinity. This would be of utmost importanceif working with real library selections involving fine affinitydiscrimination of library members.

One obvious challenge when using cell-based systems, like bacterialor yeast display (46), rather than in vitro-based ribosomaldisplay or oil-water emulsion techniques (14) is the limitedlibrary size that can be obtained due to restrictions in thetransformation step. For the staphylococcal display system describedhere, the routinely obtained transformation frequency usingpresent transformation methods would be around 10⁵ to 10⁶ transformantsper µg of DNA (10), comparable to the numbers reportedfor yeast and mammalian cell-based display systems (5). Nevertheless,one important application that obviates the need for very largelibraries is the use of the S. carnosus system in affinity maturationstrategies, where small biased libraries can be constructedon the basis of previously selected binders, thus eliminatingthe need for very large libraries (11, 25). In the future, itwould therefore be interesting to test the staphylococcal displaysystem described here for the affinity maturation of previouslyselected affibody ligands.

ACKNOWLEDGMENTS

We are grateful to George Georgiou for letting us conduct initialmodel experiments in his lab.

This work was financially supported by Cell Factory for FunctionalGenomics, a program funded by the Swedish Foundation for StrategicResearch. P.S. was supported by a postdoctoral fellowship fromthe Wenner-Gren Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden. Phone: 46 8 553 783 29. Fax: 46 8 553 784 81. E-mail: stefans{at}biotech.kth.se.

REFERENCES

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