- Email: [email protected]
[24] Production of Recombinant Human Hemoglobin A in Saccharomyces cerevisiae
374
RECOMBINANTHEMOGLOBIN
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usefully placed restriction enzyme sites. This facilitates cassette insertion of specific mutations into the globin genes and will allow analyses of structure-function relationships in hemoglobin. A comparison of the amount of total globins present in the E. coli lysate at harvest (approximately 1-5% of the total cell protein; Fig. 2) with the amount of hemoglobin present in the crude lysate (20-25/~g/ml/OD600 ; Fig. 3) indicates that most of the globin synthesized is assembled into soluble tetrameric hemoglobin. This system has been used to design and express an engineered form of human hemoglobin that has some of the properties desired in a hemoglobin-based blood substitute.ll Acknowledgments We thank CamilleMoore-Einsel and Erin Milne for their technical assistance. ~ D. L. Looker, D. Abbott-Brown, P. Cozart, S. Durfee, S. Hoffman, A. J. Mathews, J. Miller-Roehrich, S. Shoemaker, S. Trimble, G. Fermi, N. H. Komiyama, K. Nagai, and G. L. Stetler, Nature (London) 35, 258 (1992).
[24] P r o d u c t i o n o f R e c o m b i n a n t H u m a n H e m o g l o b i n A in S a c c h a r o m y c e s cerevisiae
By JiLL E. OODEN, ROY HARRIS, and MrCHAEL T. WILSON Introduction The ability to produce human hemoglobin (Hb) in a genetically engineered host microorganism offers a number of valuable opportunities. First, a recombinant microorganism provides an attractive alternative to outdated stocks of red blood ceils as a source of Hb for formulation into a Hb-based red cell substitute. Second, specific mutant Hbs can be synthesized and produced in the host for subsequent detailed structural and functional studies. Both Saccharomyces cerevisiae i-3 and Escherichia coli 4-8 have been I j. E. Ogden, J. R, Woodrow, K. A. Perks, R. Harris, D. Coghlan, and M. T. Wilson, Biomater., Artif. Cells, Immob. Biotechnol. 19, 457 (1991). 2 M. Wagenbach, K. O'Rourke, L. Vitez, A. Wieczorek, S. Hoffman, S. Durfee, J. Tedesco, and G. Stetler, Bio/Technology 9, 57 (1991). 3 D. Coghlan, G. Jones, K. A. Denton, M. T. Wilson, B. Chan, R. Harris, J. R. Woodrow, and J. E. Ogden, Eur. J. Biochem. 207, 931 (1992). 4 K. Nagai and H. C. Thergersen, Nature (London) 309, 810 (1984).
METHODS IN ENZYMOLOGY, VOL. 231
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PRODUCTION OF HEMOGLOBIN 1N YEAST
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employed for the production of recombinant human Hb. Nagai and coworkers were the first to describe the synthesis of Hb using E. coli as host. 4-6 Their approach required the separate expression of a- and /3-globins as insoluble, intracellular fusion proteins, which after solubilization and specific proteolytic cleavage were mixed in vitro with exogenous heine to produce fully functional Hb. A more straightforward method for producing recombinant Hb (rHb) in S. cerevisiae ~-3 and E. coli 7 has been described that avoids the refolding and reconstitution steps. This entails coexpression of ~- and /3-globins within the same cell. The a- and/~-globin chains fold in vivo, complete with endogenous heine, to produce soluble a2/32 tetramers. Recombinant Hb derived from E. coli using this method, however, retains the translation-initiating methionine residues at the N termini of the a- and/3-globin chains, which affects the functional properties of the molecule. 7 In contrast, recombinant Hb produced in S. cerevisiae has correctly processed globin-chain N termini 2'3 and physical and functional characterization demonstrates that the recombinant protein is identical to hemoglobin A (Hb A) purified from erythrocytes. 3 In this chapter we describe methods for expression of recombinant human Hb A (rHb A) in S. cerevisiae, the purification of the protein, and techniques used for subsequent structural and functional characterization. Coexpression of a- and fl-Globins Coexpression of a- and fl-globins to produce rHb A can be achieved either by using a single plasmid carrying both a- and fl-globin expression cassettes, or by cotransformation with two plasmids, each carrying a- and fl-globin expression cassettes and complementary auxotrophic markers. Here we describe examples of both of these approaches. Plasmids
Standard recombinant DNA techniques were used for plasmid construction. 9 The a- and fl-globin cDNAs were modified for insertion into yeast expression vectors using site-directed in vitro mutagenesis (Amer5 K. Nagai, M. F. Perutz, and C. Poyart, Proc. Natl. Acad. Sci. U.S.A. 82, 7252 (1985). 6 S. J. Hoffman and K. Nagai, U.S. Pal. 5,828,588 (1991). 7 S. I. Hoffman, D. L. Looker, J. M. Roehrick, P. E. Cozart, S. L. Durfee, J. L. Tedesco, and G. L. Stetler, Proc. Natl. Acad. Sci. U.S.A. 87, 8521 (1990). 8 D. Looker, A. J. Mathews, J. O. Neway, and G. L. Stetler, this volume [23]. 9 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Lab., Cold Spring Harbor, NY, 1989.
376
RECOMBINANT HEMOGLOBIN
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HindIIl ~ - ~ - ~ ~ C . r
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_
lobin
~_~ CYCp
2~m
pHb3
UASGALJ ~
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~
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FI6. I. Structure of plasmid pHb3. The promoter and transcription terminator sequences of CYCI (CYCp, CYCT) and PGK (PGKp, PGTT), the UAS6AL, and the orientation of the c~- and/3-globin cDNAs are shown. The E. coli ampicillin resistance gene, amp R, and S. cerevisiae LEU2d and 2/zrn plasmid functions are also indicated.
sham International, UK) and synthetic oligonucleotides (Applied Biosysterns, Foster City, CA; 38013 DNA synthesizer). ~°'1~ Coexpression Plasmid pHb3. The coexpression plasmid, pHb3 (Fig. 1), was constructed by inserting a BamHI a-globin cDNA fragment and a BglII/3-globin cDNA fragment into a pJDB207-based,12 galactose-inducible bidirectional expression vector pEK30.13 Expression of a- and /3-globin in pHb3 is directed by P G K 14 and CYCI ~5promoter fragments, respectively, under the control of the galactose-inducible upstream activation sequence of the GALI-IO genes (UASGAL). 16 This allows regulation of globin expression, so that expression is repressed in the presence of 10j. E. Ogden and C. Homer, unpublished results. ~t B. Chan, unpublished results. 12j. D. Beggs, AlfredBenzon Syrup. 16, 383 (1981). J3 E. Kenny and E. Hinchliffe, Eur. Pat. Appl. EP 317,254. ~4M. J. Dobson, M. F. Tuite, N. Roberts, A. J. Kingsman, and S. M. Kingsman, Nucleic Acids Res. 10, 2625 (1982). ~5L. Guarente and T. Mason, Cell (Cambridge, Mass.) 32, 1279 (1983). i6 M. Johnston and R. W. Davis, Mol. Cell. Biol. 4, 1440 (1984).
[24]
PRODUCTION OF HEMOGLOBIN IN YEAST
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glucose and induced when the cells are grown on galactose. Control plasmids containing only oL-globin or/3-globin cDNAs were also constructed. Plasmids for Cotransformation. pGLUo~PRB and pGT/3PRB 3 (Fig. 2) are pJDB207-based plasmids that contain the LEU2d ~2and TRP117 genes, respectively, as yeast-selectable markers. In these plasmids, appropriately modified ~- and fl-globin cDNAs are inserted downstream of the PRB1 promoter TM at a HindllI site. Similar vectors have been described previously for the expression of other heterologous proteins, is Cotransformation of pGLUoLPRB and p G T ~ P R B into leucine- and tryptophan-requiring yeast strains and subsequent growth on selective media allow for maintenance of both plasmids, p G L U ~ P R B also contains the URA3 gene ~9 as an additional selectable marker. In pGT/3PRB the ADH1 terminator is replaced by the PGK terminator.
Media Yeast minimal medium (MM) is used: 0.67% (w/v) yeast nitrogen base without amino acids (Difco, Detroit, MI), containing 2% (w/v) glucose or 2% (w/v) galactose as carbon source. Growth supplements (final concentration; uracil, 20 /xg/ml; adenine l0 /xg/ml; tryptophan, 20 gg/ml) are added when necessary.
Yeast Strains and Transformation We have used DBY745 (o~, adel, leu2, ura3) and a protease-deficient strain, DM477 (o~, pral, prbl, leu2, trpl, ura3), for the production of recombinant Hb A using pHb3. DM477 is also used for cotransformation with p G L U ~ P R B and pGT/3PRB. Plasmids are transformed into these strains using the standard spheroplast procedure of Hinnen et al., 2° selecting for complementation of the relevant auxotrophic mutation, leu2 for pHb3 and leu2 and trpl for pGLUo~PRB and pGT/3PRB cotransformants.
Small-Scale Analysis of Transformants To check for expression of a- and /3-globins, several transformants are grown on a small scale, and protein extracts are prepared and analyzed ~7K. Struhl, D. T. Stinchcomb, S. Scherer, and R. W. Davis, Gene 8, 121 (1979). ~sD. Sleep, G. P. Belfield, D. J. Ballance, J. Steven, S. Jones, L. R. Evans, P, D. Moir, and A. R. Goodey, Bio/Technology 9, 183 (1991). ~9D. Botstein, S. C. Falco, S. E. Stewart, M. Breenan, S. Scherer, D. T. Stinchcomb, K. Struhl, and R. W. Davis, Gene 8, 17 (1979), 2oA. Hinnen, J. B. Hicks, and G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 75, 1929 (1978).
378
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[24]
PRODUCTION OF HEMOGLOBIN IN YEAST
1
2
3
4
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F1G. 3. SDS-PAGE analysis of total soluble proteins from rHb A-producing yeast. Samples (100 t~g) of total soluble protein fractions from untransformed DM477 (negative control, lanes 1 and 2) and various rHb A-producing DM477 transformants (lanes 3-7) are resolved on a 15% SDS-PAGE gel with standard Hb A (2/xg) (lane 8) as control. Proteins are visualized by staining with Coomassie blue.
on S D S - P A G E gels. The ~- and/3-globins are detected either by Coomassie blue staining of the gels (Fig. 3) or by immunoblotting using a rabbit antihuman Hb antibody as probe. Methods. Transformants are grown overnight at 30° without shaking in 3 ml of MM containing glucose. Appropriate supplements are added throughout. For galactose induction, overnight cultures are diluted 100fold into 100 ml of MM containing glucose and grown at 30° with shaking until A600 n m = 1 (approximately 1 × 107 cells/ml). The cells are spun down and washed in a small volume of MM containing galactose, resuspended in 100 ml of the same medium, and grown for a further 48 hr. Overnight cultures of DM477 cotransformants are inoculated into 100 ml of MM containing glucose and grown for 72 hr at 30° with shaking. The cells are harvested in 50-ml aliquots, centrifuged, and washed with 10 ml of 150 mM HEPES, pH 7.0. The cell pellets are finally resuspended in 1.5 ml of the same buffer, and glass beads (0.4 mm diameter, BDH) are added up to the meniscus. The cells are lysed by vortexing for 4 × 30 sec, with intermittent cooling on ice. A further 0.5 ml of 150 mM HEPES, pH 7.0, is added and the suspension is vortexed for 30 sec. After allowing the glass beads to settle, the cell extract is transferred to a clean Eppendorf tube and centrifuged for 5 rain. This separates the soluble protein fractions (supernatant) from the insoluble fractions (pellet). Samples of the soluble
380
RECOMBINANTHEMOGLOBIN
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protein fractions (50-I00/xg total protein) and standard Hb A (200 ng-2 /xg, Sigma) in sample buffer [10 mM Tris-HCl, pH 8.0, 2% (w/v) SDS, 5% (w/v) 2-mercaptoethanol, 1 mM EDTA, 0.01% bromphenol blue] are boiled immediately in a water bath and then resolved on 15% reducing SDS-PAGE gels. We use gels of approximately 15 x 18 cm to ensure good separation of the globin chains. Proteins can be detected by staining the gels with Coomassie blue (Fig. 3). Alternatively, proteins can be electroblotted onto Immobilon P membrane (Millipore, Bedford, MA). Nonspecific binding sites on the membrane are blocked by washing in TBS (20 mM Tris-HC1, pH 7.6, 0.15 M NaCI) containing 0.05% (v/v) Nonidet P-40 and 5% (w/v) milk powder for 45 rain. The membrane is incubated with rabbit antihuman Hb antibody (1 : 1000 dilution; Sigma) in TBS containing 0.05% Nonidet P-40 and 0.2% milk powder for 1 hr, washed for 3 × 5 min in the same buffer, and then incubated with peroxidase-conjugated goat antirabbit antibody (1 : 1000 dilution; Sigma) for 1 hr. After washing for 3 × 5 min, immunoreactive proteins are visualized using the ECL detection system (Amersham), according to the manufacturer's instructions.
Assays of Hemoglobin in Cell Extracts There can be considerable variation in levels of rHb A expression between transformants, probably due to differences in plasmid copy number. It is advisable, therefore, to screen several transformants before choosing one for large-scale growth and subsequent purification of rHb A. Analysis of transformants by SDS-PAGE gels gives some indication of differences in expression levels. We also use a spectrophotometric assay in which rHb A in cell extracts is quantitated from the height of the Soret peak in second-derivative spectra, by comparison with standard Hb A of known concentration. 21 It is important to use samples that have been deoxygenated with sodium dithionite to distinguish rHb A in cell extracts 0tmax 430 nm) from the indigenous yeast cytochrome c (~max 415 nm). Because cell extracts can be quite turbid, we find that clarification using polyethyleneimine (PEI) prior to measuring spectra gives better resuits. Methods
PEI [20/,1 of 5% (v/v) solution in 150 mM HEPES, pH 7.0] is added to l ml of cell extract in an Eppendorf tube and the mixture is centrifuged 2I H. van Urk and D. Coghlan, unpublished results.
[24]
PRODUCTION OF HEMOGLOBIN IN YEAST
381
for 30 sec. The clarified supernatant is removed and 12.5 t~l of sodium dithionite [10% (w/v) solution in 150 mM HEPES, pH 7.0] is added. The absorbance spectrum (290-610 rim) of the sample is measured in the second-derivative mode. Dilutions of standard Hb A, treated in the same way, are used to prepare a standard curve. The rHb A content of cell extracts is calculated as a percentage of total soluble protein, determined using Pierce (Rockford, IL) Coomassie protein assay reagent. Purification of rHb A We use cation-exchange chromatography for purifying rHb A from yeast cell lysates. To prevent the formation of metHb during purification, all chromatography buffers are purged thoroughly with helium to displace dissolved oxygen. In addition CO is bubbled through the lysate for 3-5 rain immediately after cell breakage. The rHb A fractions are maintained in the carbonmonoxy form by repurging with CO at each stage of purification. CarbonmonoxyHb can be converted to the oxy form by flushing the solution on ice with oxygen under bright illumination.
Large-Scale Growth of Yeasts and Preparation of Cell Lysates Yeast transformants can be grown on a large scale in shake flasks (12 x 2-liter flasks each containing I liter of medium). We also grow yeasts by fed batch fermentation using established protocols.Z-~ For purification of rHb A from DBY745 transformants it is important to incorporate protease inhibitors [2 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM EDTA] in the cell lysis buffer to prevent protease damage of rHb A. This is not necessary when using DM477. Methods. A suitable transformant is grown overnight in 200 ml of MM containing 2% (w/v) glucose at 30° with shaking. Growth supplements are added throughout as necessary. The overnight culture is diluted 1 : 100 into 12 x l-liter batches of MM containing 2% (w/v) galactose (for pHb3 transformants) or MM containing 2% (w/v) glucose (for DM477 cotransformants). Cultures are grown at 30° with shaking until in the stationary phase of growth (usually 72 hr). Yeast cells are harvested by centrifugation at 7000 g for 20 rain at 4°. The cells are either used immediately or can be frozen at - 7 0 ° in thin layers in plastic bags for storage at - 20°. The yeast cells are resuspended in 10 mM phosphate buffer, pH 6.5 (containing 2 mM PMSF, 2 mM EDTA if necessary), at a concentration of approximately 60% wet weight/volume 22 S. H. Collins, in "Protein Production by Biotechnology" (T. J, R. Harris, ed.), p. 61. Elsevier, New York, 1990.
382
RECOMBINANTHEMOGLOBIN
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(100 g of cells made up to ~160 ml with buffer). The cells are broken in a Bead Beater (Biospec Products, Bartlesville, OK) using 0.4-ram-diameter glass beads and ice cooling for four 30-sec periods, each separated by a 4-rain interval. The glass beads are removed by vacuum filtration through a glass scinter funnel, and the filtrate is clarified by centrifugation at 30,000 g for 30 rain at 4°. The supernatant is removed, taking care not to disturb the surface lipid layer, and is immediately purged with CO for 5 rain. The extract is kept on ice throughout. The CO-purged lysate is desalted using a Sephadex G-25 (Pharmacia, Piscataway, NJ) column (5 x 30 cm; -~600 ml) equilibrated at 4° with l0 mM phosphate buffer, pH 6.5. The column eluate is monitored at A28o,m and the main protein fraction is collected. The pH of the desalted eluate is adjusted if necessary to pH 6.5 with glacial acetic acid. The conductivity of the eluate should be reduced, if necessary, to less than 2 millisieverts by adding Milli Q water. The eluate is repurged briefly with CO before the next step in purification.
Ion-Exchange Chromatography Desalted eluates are subjected to three successive ion-exchange chromatography steps23: S-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Q Sepharose Fast Flow. Methods. The desalted extract is applied to a 50-ml (3.5 x 5 cm) S-Sepharose Fast Flow column (Pharmacia) equilibrated at 4° with I0 mM phosphate buffer, pH 6.5, at a flow rate of 2.5 ml/min. The rHb A is eluted using a linear pH gradient to pH 8.0 (10 mM phosphate) over 10 column volumes using a Pharmacia FPLC chromatography system. The buffers are purged with helium before use. The elution of rHb A (red fractions) is monitored at A280nmand A405,m. The pooled fractions containing rHb A are purged with CO and the pH is reduced to 6.5 by the addition of glacial acetic acid. The S eluate (pH 6.5) is loaded onto a 10-ml (1.2 x 9 cm) CM-Sepharose Fast Flow column (Pharmacia) at 4 ° equilibrated in 10 mM phosphate, pH 6.5, at a flow rate of 1 ml/min. The same elution procedure (linear pH gradient up to pH 8.0) is used as described here. The rHb A elutes as a single major A405 nm peak, and fractions collected are pooled. A 10-ml Q-Sepharose Fast Flow (FF) column (Pharmacia) (1,5 × 6 cm) is equilibrated in 20 mM Tris-HC1, pH 8.5, at 4° and a flow rate of l ml/min. The pH of the CM eluate is adjusted to 8.5 by the addition of 23 D. Coghlan, unpublished results.
[24]
PRODUCTION OF HEMOGLOBIN IN YEAST
1
2
3
4
383
5
FIG. 4. SDS-PAGE analysis of rHb A purification fractions. Samples are resolved on a 20% homogeneous Phast gel (Pharmacia) and stained with Coomassie blue. All lanes, except lane 2, are loaded with a total of I ~g Hb. Lane 1, standard Hb A (Sigma); lane 2, yeast cell lysate (2 ~g total protein); lane 3, S-Sepharose eluate; lane 4, CM-Sepharose eluate; and lane 5, Q-Sepharose eluate. On these gels a- and/3-globins comigrate.
2 M Tris base and is repurged with CO. This is then loaded onto the Q - S e p h a r o s e FF column at 1 ml/min. The flow rate is reduced to 0.5 ml/ rain or less, and the r H b A is eluted stepwise using either 20 m M bis-Tris buffer, p H 6.6, or 20 m M H E P E S , p H 6.6. This step not only gives an i m p r o v e m e n t in purity but also allows buffer exchange and is an important concentration step.
Purity Assessment The purity of r H b A is monitored during the purification either by s p e c t r o p h o t o m e t r y or by using reducing S D S - P A G E . A rapid quantitative estimation of purity can be obtained f r o m UV/Vis scans (250-650 nm) of r H b A fractions and standard Hb A in their c a r b o n m o n o x y forms, using the following c o m p a r a t i v e a b s o r b a n c e ratios: (A419/Az76)rHb A/(A419/ A276)Hb A. F o r S D S - P A G E , fractions containing r H b A at a concentration of approximately 1 mg/ml in sample buffer (see above) are resolved either on 20% h o m o g e n e o u s Phast gels (Pharmacia) (Fig. 4) or on conventional S D S - P A G E gels.
384
RECOMBINANT HEMOGLOBIN
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Techniques for Physical Characterization of rHb A
Methods HPLC Separation of Globin Chains. A 100-/zl sample of purified rHb A (50-500/~g) is mixed with 100/xl of 8 M urea and chromatographed on an HPLC Vydac C4 reverse-phase (RP) column. 24 The column is equilibrated at 1 ml/min in 70% solvent A:30% solvent B [solvent A is 0.1% trifluoroacetic acid (TFA) in water; solvent B is 90% acetonitrile/0.09% TFA]. Solvent B is increased to 45% over 5 rain, followed by a 30-rain linear gradient to 55% B. Separation of heine,/3-globin, and c~-globin is monitored at A214 nm and A280 nm" Peptide Mapping. Samples (200-500 t~g rHb A or isolated a- or /3-globin chains) in 500/zl of buffer are precipitated in an Eppendorf tube by adding 1 ml of acetone. After vortexing, the tubes are incubated at - 20° for I hr and then centrifuged for 10 rain. The supernatant is discarded; the pellets are dried briefly by rotary evaporation and are then resuspended in 100/A of 6 M guanidine hydrochloride in 0.5 M Tris-HCl, pH 8.0, 2 mM EDTA. The samples are reduced [10/zl of 100 mM dithiothreitol (DTT) in water, 37° for 1-2 hr], carboxyamidomethylated [20 tzl of 100 mM iodoacetamide (Sigma) in 0.1 M Tris-HCl, pH 8.0, 37° for 2-3 hr in the dark], and then diluted 1 : 3 by adding 400/zl of water. Samples are digested with 3 × 5-/.d aliquots of trypsin (Sigma, 1 mg/ml in 1 mM HC1, stored at 4 °) over 48 hr at 37° with shaking. The digests are separated on a Pharmacia Pep-S RP-column. The column is equilibrated in 95% solvent A : 5% solvent B (solvent A is 0.1% TFA in water; solvent B is 70% acetonitrile/ 0.085% TFA) at 0.5 ml/min. The peptides are eluted using a linear gradient up to 60% B over 50 min with detection at A214o~. Amino Acid and N-Terminal Analysis. The a- and/3-globin chains of rHb A and Hb A are separated by RP-HPLC as described above. Aliquots containing 20/zg of each chain are dried separately and resuspended in 20 tzl of 0.1% TFA. Each chain (15 t~g) is subjected to 10 cycles of automated Edman degradation using an Applied Biosystems 477A protein sequencer and to automated acid hydrolysis and amino acid composition analysis using an Applied Biosystems 420 A amino acid analyzer. Mass Spectrometric Analysis. Purified rHb A (I rag) is dialyzed against Milli Q water and analyzed by Electrospray mass spectrometry (ESMS) using a VG Biotech BIO-Q mass spectrometer (M-Scan Ltd., Ascot, UK). 24 S. Rahbar and Y. A s m e r o m ,
Hemoglobin 13, 475 (1989).
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PRODUCTION OF HEMOGLOBIN IN YEAST
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Functional Studies The simplest and most direct methods to confirm that purified yeastderived rHb A is fully functional rely on optical absorption spectroscopy. We have used such an approach to determine the oxygen equilibrium curves and CO combination kinetics for the recombinant protein. These methods have been described extensively elsewhere. 2-s'26 Only minimal details of the most straightforward techniques will be described here. Prior to functional studies, rHb A purified in the carbonmonoxy form is converted to the oxy form by exposure on ice to a stream of humidified pure oxygen under illumination (Osram Dulux EL.20W low-heat bulb) with periodic agitation for approximately 1 hr. Control experiments are carried out with standard Hb A purified from erythrocytes using established procedures. 27
Oxygen Equilibrium Curves Oxygen equilibrium curves may be obtained conveniently without the requirement for specialized apparatus either by (1) serial addition of air to a modified optical cuvette containing a d e o x y H b solution under vacuum or by (2) controlled enzymatic deoxygenation of a solution of o x y H b in an optical cuvette equipped with an oxygen electrode. Titration Method, The titration method has been described adequately elsewhere. 26 This method is simple to use providing that care is taken to avoid excessive bubble formation and hence denaturation during the evacuation procedures needed to generate d e o x y H b , and that full equilibration is achieved after each addition of air. Equilibration is ensured by transfer of the sample to the tonometer bulb (Fig. 5) by tilting the apparatus, which is then agitated in a water bath for 5 rain. An example of an oxygen equilibrium curve obtained for rHb A using this method is given in Fig. 6. Enzymatic Method. Enzymatic depletion of oxygen has the advantage that Hb is not subjected to lengthy evacuation procedures and also that the cuvette remains at all times within the spectrophotometer housing, thus eliminating repositioning errors. The disadvantages are that the substrates and/or products of the enzymes used to consume oxygen may degrade Hb. We have found that a suitable method uses cytochrome c oxidase zs E. Antonini and M. Brunori, in "Hemoglobin and Myoglobin in Their Reactions with Ligands, Frontiers in Biology" (A. Neuberger and E. L. Tatum, eds.). North-Holland Publ., Amsterdam, 1971. 26 B. Giardina and G. Amiconi, this series, Vol. 76, p. 417. 27A. Riggs, this series, Vol. 76, p. 5.
386
RECOMBINANT HEMOGLOBIN
vacuum pump
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FIG. 5. Diagram of tonometer bulb used to determine oxygen equilibrium curves. The total combined volume (approximately 150 ml) of the tonometer and cuvette must be determined by weighing the system empty and when completely filled with a liquid of known density. A sample of hemoglobin is deoxygenated by evacuation, and the deoxy spectrum is taken, The volume of the sample following deoxygenation is similarly determined by weight. Additions of 0.5-2 ml of air (21% oxygen) at known atmospheric pressure and humidity are made via the vaccine cap as shown. Tonometers may be modified for use with small volumes. 25
and ascorbate/cytochrome c. Alternatively, ascorbate oxidase/ascorbate may be used. These systems reduce oxygen to water as opposed to hydrogen peroxide. It is important to remove the oxygen in about 20 min to minimize the risk of ascorbate damage to the heine group. 28 Methods. A simple system comprises a normal 1-cm pathlength optical cuvette, with ground aperture, into which an oxygen electrode (Yellow Springs Instrument Company, Yellow Springs, OH) can fit tightly. A small gap should remain at one point through which the reactants can be injected using a microsyringe. It is essential that the electrode protrudes into the cuvette and is close (1 cm) to a magnetic follower at the bottom of the cuvette, because the response of the electrode is critically dependent on efficient stirring of the solution close to the membrane. A suitable system 28 j. C. Docherty and S. B. Brown, Biochem. J. 207, 583 (1982).
[24]
387
PRODUCTION OF HEMOGLOBIN IN YEAST
0.3 1.0 0.2 Y 0.5 0,1
0 500
0
550 Wavelength (rim)
600
I
0
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10 Oxygen pressure (mm Hg)
I
20
FIG. 6. Determination of o x y g e n equilibrium curves. (a) Spectral transition from deoxyto o x y r H b A (25 ~ M h e m e in 30 m M H E P E S , pH 7.0, 0.2 M NaCI, 25 °) (a --~ k) on titration with air. The final s p e c t r u m was obtained under pure oxygen. The spectra are characteristic of authentic h u m a n Hb A. (b) The fractional saturation (Y) determined from ,£4s76_560,m is plotted against o x y g e n pressure ( m m Hg). The equilibrium curve is characterized by a Ps0 (the partial pressure at which Hb is 50% saturated) of 9.0 m m Hg, nm~~ = 2.9.
comprises Hb at a concentration of 25-100 /xM heine, I0 mM sodium ascorbate, 50/,M tetramethylphenylenediamine (TMPD; electron mediator, Sigma), 0.25/,M cytochrome c (Type VI, Sigma), and 0.5/,M (total heine) bovine cytochrome c oxidase (prepared according to Yonetani 29) in 0.1 M sodium phosphate buffer, pH 6.8, containing 0.05% (v/v) Tween 80, a nonionic detergent to solubilize the enzyme. Alternatively, 2 units ascorbate oxidase (Sigma) can be substituted for cytochrome c oxidase, and the TMPD, cytochrome c, and Tween 80 can be omitted. In both systems the enzyme should be sufficiently active to deoxygenate the solution within about 20 rain. Oxygen equilibrium curves are obtained by plotting the absorbance at a single wavelength (e.g., 576 nm) against the calibrated output from the oxygen electrode. Alternatively, spectra may be recorded throughout oxygen depletion as in Fig. 6. These must, however, be taken at rapid scan rates to ensure minimal distortion due to oxygen depletion during the course of each scan) ° 29 T. Yonetani, J. Biol. Chem. 236, 1680 (1961). 3o K. D. Vandegriff and R, I. Shrager, this series, Vol. 232.
388
RECOMBINANT HEMOGLOBIN
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Carbon Monoxide Binding Kinetics The autocatalytic time course for CO binding to Hb is a convenient probe for the T to R transition and hence functional integrity. The kinetics of binding may be followed at low protein concentration because d e o x y H b tetramers are stable, and dissociation into dimers does not occur in the micromolar concentration range. 3j Methods'. O x y H b (5 to I0 ml of approximately 5/~M heine in 0.1 M phosphate buffer, pH 7.0) solution is partially deoxygenated by three cycles of evacuation and flushing with nitrogen and then drawn into a syringe. Full deoxygenation is achieved by addition of a few grains of solid sodium dithionite or by addition of a 200 mM solution of sodium dithionite made up in anaerobic buffer to a final concentration of 2 raM. This solution is mixed in a standard stopped-flow apparatus (Applied Photophysics, Leatherhead, U K) with solutions of CO in buffer containing 2 mM sodium dithionite. Solutions of CO, at required concentrations (e.g., 25-200 p~M), are prepared by diluting a saturated solution of CO in water (equilibration of water at 20 ° with CO at atmospheric pressure (101 kPa) gives a 1.03 mM solution of CO). Rapid-mixing devices designed to be used with spectrophotometers (e,g., HiTech, Salisbury, UK) are also suitable for use at low CO concentrations. In this case it is often necessary, because of the slow response of the A/D converter, to use an analog signal from the spectrophotometer both for display on an oscilloscope and for conversion to digital form for analysis. Absorbance changes at 436 nm are recorded as a function of time in the l-msec to l-sec time range. At pH 7.0 in 0.1 M phosphate buffer at 20 °, the time course for CO combination to d e o x y H b is autocatalytic, reflecting the T to R transition on CO binding. A series of time courses over a range of CO concentrations allow the second-order rate constant (l') to be determined by standard procedures. 25 The rate constant under these conditions increases from approximately 1 × 10~ M -1 sec J to 2 × 105 M -1 sec -1 throughout the course of binding (see Table I). The time course for CO combination to rHb A should be free of any fast initial component (/' > 106 M -1 s e c - t ) , indicating the presence of free or noncooperative subunits.
Results and Comments Using the purification methods described, we routinely obtain rHb A of >95% purity, with an overall recovery of 50% even when starting with yeast transformants producing rHb A at as little as 0.5% of the total 31 B. W. Turner, D. W. Pettigrew, and G. Ackers, this series, Vol. 76, p. 596.
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PRODUCTION OF HEMOGLOBIN IN YEAST
TABLE 1 PHYSICAL AND FUNCTIONAL CHARACTERISTICS OF YEAST-DERIVED STANDARD H b A FROM ERYTHROCYTES
Parameter Spectroscopic analysis
RP-HPLC analysis Peptide mapping N-terminal analysis Amino acid analysis Electrospray mass spectrometry
Oxygen binding properties
CO binding properties
rHb A
AND
Characteristics Normalized spectra of the oxy, deoxy, and carbonmonoxy derivatives of rHb A and Hb A are superimposable between 350 and 650 nm Stoichiometry of c~-globin:/3-globin : heine ratio is identical to that of Hb A Tryptic maps of rHb A and recombinant c~- and/3globin chains are identical to those of Hb A Recombinant c~- and/3-globin chains have correctly processed N termini Amino acid composition of rHb A is identical to standard Hb A Mass: ~-globin, 15,126.0 Da;/3-globin, 15,866.2 Da. These masses are within 1 Da of those of standard c~- and/3-globins Ps0: rHb A, 9.0 mm Hg; Hb A, 8.9 mm Hg (25 tzM heine, 30 mM HEPES, pH 7.0, 0.2 M NaCI, 25°) nn~a~: rHb A, 2.9; Hb A, 3.0 Bohr effect [A log Pso/A pH (pH 7.0 - 7.6)]: rHb A, -0.37; Hb A, -0.34 l ' : r H b A , 2.19 x 0.05 ± l0 sM l s e c - ~ ; H b A , 2.02 x 0.03 +- l05 M -t sec -t (2/xM heine. 0.1 M sodium phosphate, pit 7.0, 20°)
s o l u b l e cell p r o t e i n . R e p r e s e n t a t i v e r e s u l t s for t h e p h y s i c a l a n d f u n c t i o n a l c h a r a c t e r i z a t i o n o f r H b A e m p l o y i n g t h e t e c h n i q u e s o u t l i n e d a r e g i v e n in T a b l e I. P h y s i c a l a n a l y s i s d e m o n s t r a t e s t h a t r H b A is i d e n t i c a l to H b A purified from erythrocytes. The spectroscopic and chromatographic techn i q u e s a l s o c o n f i r m t h a t t h e y e a s t - d e r i v e d p r o t e i n c o n t a i n s a full c o m p l e m e n t o f e n d o g e n o u s l y p r o d u c e d h e m e g r o u p s (i.e., f o u r h e i n e g r o u p s p e r tetramer), In addition, equilibrium and kinetic measurements of the oxygen a n d C O b i n d i n g p r o p e r t i e s o f r H b A d e m o n s t r a t e t h a t the f u n c t i o n a l c h a r a c t e r i s t i c s o f t h e r e c o m b i n a n t p r o t e i n a r e i n d i s t i n g u i s h a b l e f r o m H b A. T h e a b i l i t y to s y n t h e s i z e a u t h e n t i c h u m a n H b A in a s i m p l e e u k a r y o t e , s u c h as S. cerevisiae, p r o v i d e s an i d e a l s y s t e m for t h e e x p r e s s i o n o f engineered mutant Hbs for structural and functional studies. The coexpression systems and purification protocol described here can be easily a d a p t e d for t h e p r o d u c t i o n o f m u t a n t H b s for f u r t h e r a n a l y s i s . M o r e o v e r , genetically engineered yeast producing recombinant human Hb A or mut a n t H b s offers t h e o p p o r t u n i t y f o r p r o v i s i o n o f H b for d e v e l o p m e n t into Hb-based oxygen carriers.
390
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Acknowledgments We would like to thank many of our colleagues for helpful discussion during the preparation of this manuscript, and especially Bernard Chan, Sarah Gilbert, Katharine Denton, Gareth Jones, David Coghlan, and Henk van Urk for allowing us to refer to their unpublished data. We also thank Kelly Morris for preparing the manuscript.
[25] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f R e c o m b i n a n t H u m a n S i c k l e H e m o g l o b i n E x p r e s s e d in Y e a s t
By
JOSE JAV1ER M A R T I N DE L L A N O , O L A F S C H N E E W I N D , GARY L. STETLER, and JAMES M. MANNING
In the genetic disease sickle cell anemia the abnormal hemoglobin S (Hb S) that is expressed has the hydrophobic side chain of Val-6(/3) in place of the hydrophilic side chain of Glu-6(/3) in hemoglobin A. This substitution on the exterior of the protein causes the aggregation of deoxygenated hemoglobin S tetramers, leading to the distortion of erythrocytes in the venous circulation. I-8 Although the initial aggregation contact is between Val-6(/3) and the Phe-85(/3)/Leu-88(/3) region of an adjacent Hb S tetramer, there are many subsequent points of contact that strengthen the overall stability of the polymer at low oxygen tensions. 3-5 Although the identity of some of the contact sites in the aggregate is known, 3-5'9 there is a lack of information about other contract sites as well as their relative contribution to the overall strength of the aggregate. The number and nature of such susceptible sites have been limited mainly to those hydrophilic side chains with enhanced reactivity because of their location in the protein. 8'~° The availability of recombinant DNA technology now permits studies at any site on the hemoglobin S tetramer, heretofore not possible to modify by other methods. For hemoglobin S, the ability to alter hydrophobic sites 1 L. Pauling, H. Itano, S. J. Singer, and I. C. Wells, Science 110, 543 (1949). 2 V. M. Ingram, Nature (London) 178, 792 (1956). 3 B. C. Wishner, K. B. Ward, E. E. Lattman, and W. E, Love, J. Mol. Biol. 98, 179 (1975). 4 S. J. Edelstein and R. H. Crepeau, J. Mol. Biol. 134, 851 (1979). 5 T. E. Wellems and R. Josephs, J, Mol. Biol. 135, 651 (1979). 6 j, B. Herrick, Arch. Intern. Med. 6, 517 (1910). 7 A. Cerami and J. M. Manning, Proc. Natl. Acad. Sci, U.S.A. 68, 1180 (1971). 8 j. Dean and A. N. Schechter, N. Engl. J. Med. 299, 752, 804, 863 (1978). 9 R. M. Bookchin, R. L. Nagel, and H. M. Ranney, J. Biol. Chem. 242, 248 (1967). t0 j. M. Manning, Adv. Enzymol. Mol, Biol. 64, 55 (1991).
METHODS IN ENZYMOLOGY,VOL. 231
Copyright ~) 1994by AcademicPress, lnc. All rightsof reproductionin any form reserved.
RECOMBINANTHEMOGLOBIN
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usefully placed restriction enzyme sites. This facilitates cassette insertion of specific mutations into the globin genes and will allow analyses of structure-function relationships in hemoglobin. A comparison of the amount of total globins present in the E. coli lysate at harvest (approximately 1-5% of the total cell protein; Fig. 2) with the amount of hemoglobin present in the crude lysate (20-25/~g/ml/OD600 ; Fig. 3) indicates that most of the globin synthesized is assembled into soluble tetrameric hemoglobin. This system has been used to design and express an engineered form of human hemoglobin that has some of the properties desired in a hemoglobin-based blood substitute.ll Acknowledgments We thank CamilleMoore-Einsel and Erin Milne for their technical assistance. ~ D. L. Looker, D. Abbott-Brown, P. Cozart, S. Durfee, S. Hoffman, A. J. Mathews, J. Miller-Roehrich, S. Shoemaker, S. Trimble, G. Fermi, N. H. Komiyama, K. Nagai, and G. L. Stetler, Nature (London) 35, 258 (1992).
[24] P r o d u c t i o n o f R e c o m b i n a n t H u m a n H e m o g l o b i n A in S a c c h a r o m y c e s cerevisiae
By JiLL E. OODEN, ROY HARRIS, and MrCHAEL T. WILSON Introduction The ability to produce human hemoglobin (Hb) in a genetically engineered host microorganism offers a number of valuable opportunities. First, a recombinant microorganism provides an attractive alternative to outdated stocks of red blood ceils as a source of Hb for formulation into a Hb-based red cell substitute. Second, specific mutant Hbs can be synthesized and produced in the host for subsequent detailed structural and functional studies. Both Saccharomyces cerevisiae i-3 and Escherichia coli 4-8 have been I j. E. Ogden, J. R, Woodrow, K. A. Perks, R. Harris, D. Coghlan, and M. T. Wilson, Biomater., Artif. Cells, Immob. Biotechnol. 19, 457 (1991). 2 M. Wagenbach, K. O'Rourke, L. Vitez, A. Wieczorek, S. Hoffman, S. Durfee, J. Tedesco, and G. Stetler, Bio/Technology 9, 57 (1991). 3 D. Coghlan, G. Jones, K. A. Denton, M. T. Wilson, B. Chan, R. Harris, J. R. Woodrow, and J. E. Ogden, Eur. J. Biochem. 207, 931 (1992). 4 K. Nagai and H. C. Thergersen, Nature (London) 309, 810 (1984).
METHODS IN ENZYMOLOGY, VOL. 231
Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
[24]
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employed for the production of recombinant human Hb. Nagai and coworkers were the first to describe the synthesis of Hb using E. coli as host. 4-6 Their approach required the separate expression of a- and /3-globins as insoluble, intracellular fusion proteins, which after solubilization and specific proteolytic cleavage were mixed in vitro with exogenous heine to produce fully functional Hb. A more straightforward method for producing recombinant Hb (rHb) in S. cerevisiae ~-3 and E. coli 7 has been described that avoids the refolding and reconstitution steps. This entails coexpression of ~- and /3-globins within the same cell. The a- and/~-globin chains fold in vivo, complete with endogenous heine, to produce soluble a2/32 tetramers. Recombinant Hb derived from E. coli using this method, however, retains the translation-initiating methionine residues at the N termini of the a- and/3-globin chains, which affects the functional properties of the molecule. 7 In contrast, recombinant Hb produced in S. cerevisiae has correctly processed globin-chain N termini 2'3 and physical and functional characterization demonstrates that the recombinant protein is identical to hemoglobin A (Hb A) purified from erythrocytes. 3 In this chapter we describe methods for expression of recombinant human Hb A (rHb A) in S. cerevisiae, the purification of the protein, and techniques used for subsequent structural and functional characterization. Coexpression of a- and fl-Globins Coexpression of a- and fl-globins to produce rHb A can be achieved either by using a single plasmid carrying both a- and fl-globin expression cassettes, or by cotransformation with two plasmids, each carrying a- and fl-globin expression cassettes and complementary auxotrophic markers. Here we describe examples of both of these approaches. Plasmids
Standard recombinant DNA techniques were used for plasmid construction. 9 The a- and fl-globin cDNAs were modified for insertion into yeast expression vectors using site-directed in vitro mutagenesis (Amer5 K. Nagai, M. F. Perutz, and C. Poyart, Proc. Natl. Acad. Sci. U.S.A. 82, 7252 (1985). 6 S. J. Hoffman and K. Nagai, U.S. Pal. 5,828,588 (1991). 7 S. I. Hoffman, D. L. Looker, J. M. Roehrick, P. E. Cozart, S. L. Durfee, J. L. Tedesco, and G. L. Stetler, Proc. Natl. Acad. Sci. U.S.A. 87, 8521 (1990). 8 D. Looker, A. J. Mathews, J. O. Neway, and G. L. Stetler, this volume [23]. 9 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Lab., Cold Spring Harbor, NY, 1989.
376
RECOMBINANT HEMOGLOBIN
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HindIIl ~ - ~ - ~ ~ C . r
~x~g
_
lobin
~_~ CYCp
2~m
pHb3
UASGALJ ~
Hindlll" ~__R
~
PGKv
Sail
FI6. I. Structure of plasmid pHb3. The promoter and transcription terminator sequences of CYCI (CYCp, CYCT) and PGK (PGKp, PGTT), the UAS6AL, and the orientation of the c~- and/3-globin cDNAs are shown. The E. coli ampicillin resistance gene, amp R, and S. cerevisiae LEU2d and 2/zrn plasmid functions are also indicated.
sham International, UK) and synthetic oligonucleotides (Applied Biosysterns, Foster City, CA; 38013 DNA synthesizer). ~°'1~ Coexpression Plasmid pHb3. The coexpression plasmid, pHb3 (Fig. 1), was constructed by inserting a BamHI a-globin cDNA fragment and a BglII/3-globin cDNA fragment into a pJDB207-based,12 galactose-inducible bidirectional expression vector pEK30.13 Expression of a- and /3-globin in pHb3 is directed by P G K 14 and CYCI ~5promoter fragments, respectively, under the control of the galactose-inducible upstream activation sequence of the GALI-IO genes (UASGAL). 16 This allows regulation of globin expression, so that expression is repressed in the presence of 10j. E. Ogden and C. Homer, unpublished results. ~t B. Chan, unpublished results. 12j. D. Beggs, AlfredBenzon Syrup. 16, 383 (1981). J3 E. Kenny and E. Hinchliffe, Eur. Pat. Appl. EP 317,254. ~4M. J. Dobson, M. F. Tuite, N. Roberts, A. J. Kingsman, and S. M. Kingsman, Nucleic Acids Res. 10, 2625 (1982). ~5L. Guarente and T. Mason, Cell (Cambridge, Mass.) 32, 1279 (1983). i6 M. Johnston and R. W. Davis, Mol. Cell. Biol. 4, 1440 (1984).
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PRODUCTION OF HEMOGLOBIN IN YEAST
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glucose and induced when the cells are grown on galactose. Control plasmids containing only oL-globin or/3-globin cDNAs were also constructed. Plasmids for Cotransformation. pGLUo~PRB and pGT/3PRB 3 (Fig. 2) are pJDB207-based plasmids that contain the LEU2d ~2and TRP117 genes, respectively, as yeast-selectable markers. In these plasmids, appropriately modified ~- and fl-globin cDNAs are inserted downstream of the PRB1 promoter TM at a HindllI site. Similar vectors have been described previously for the expression of other heterologous proteins, is Cotransformation of pGLUoLPRB and p G T ~ P R B into leucine- and tryptophan-requiring yeast strains and subsequent growth on selective media allow for maintenance of both plasmids, p G L U ~ P R B also contains the URA3 gene ~9 as an additional selectable marker. In pGT/3PRB the ADH1 terminator is replaced by the PGK terminator.
Media Yeast minimal medium (MM) is used: 0.67% (w/v) yeast nitrogen base without amino acids (Difco, Detroit, MI), containing 2% (w/v) glucose or 2% (w/v) galactose as carbon source. Growth supplements (final concentration; uracil, 20 /xg/ml; adenine l0 /xg/ml; tryptophan, 20 gg/ml) are added when necessary.
Yeast Strains and Transformation We have used DBY745 (o~, adel, leu2, ura3) and a protease-deficient strain, DM477 (o~, pral, prbl, leu2, trpl, ura3), for the production of recombinant Hb A using pHb3. DM477 is also used for cotransformation with p G L U ~ P R B and pGT/3PRB. Plasmids are transformed into these strains using the standard spheroplast procedure of Hinnen et al., 2° selecting for complementation of the relevant auxotrophic mutation, leu2 for pHb3 and leu2 and trpl for pGLUo~PRB and pGT/3PRB cotransformants.
Small-Scale Analysis of Transformants To check for expression of a- and /3-globins, several transformants are grown on a small scale, and protein extracts are prepared and analyzed ~7K. Struhl, D. T. Stinchcomb, S. Scherer, and R. W. Davis, Gene 8, 121 (1979). ~sD. Sleep, G. P. Belfield, D. J. Ballance, J. Steven, S. Jones, L. R. Evans, P, D. Moir, and A. R. Goodey, Bio/Technology 9, 183 (1991). ~9D. Botstein, S. C. Falco, S. E. Stewart, M. Breenan, S. Scherer, D. T. Stinchcomb, K. Struhl, and R. W. Davis, Gene 8, 17 (1979), 2oA. Hinnen, J. B. Hicks, and G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 75, 1929 (1978).
378
RECOMBINANT HEMOGLOBIN
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qJ
-o~E
~.
_
e"
I
~o~ °~
0
u)
---
~.~
~
EM Q
~
[24]
PRODUCTION OF HEMOGLOBIN IN YEAST
1
2
3
4
5
6
7
379
8
F1G. 3. SDS-PAGE analysis of total soluble proteins from rHb A-producing yeast. Samples (100 t~g) of total soluble protein fractions from untransformed DM477 (negative control, lanes 1 and 2) and various rHb A-producing DM477 transformants (lanes 3-7) are resolved on a 15% SDS-PAGE gel with standard Hb A (2/xg) (lane 8) as control. Proteins are visualized by staining with Coomassie blue.
on S D S - P A G E gels. The ~- and/3-globins are detected either by Coomassie blue staining of the gels (Fig. 3) or by immunoblotting using a rabbit antihuman Hb antibody as probe. Methods. Transformants are grown overnight at 30° without shaking in 3 ml of MM containing glucose. Appropriate supplements are added throughout. For galactose induction, overnight cultures are diluted 100fold into 100 ml of MM containing glucose and grown at 30° with shaking until A600 n m = 1 (approximately 1 × 107 cells/ml). The cells are spun down and washed in a small volume of MM containing galactose, resuspended in 100 ml of the same medium, and grown for a further 48 hr. Overnight cultures of DM477 cotransformants are inoculated into 100 ml of MM containing glucose and grown for 72 hr at 30° with shaking. The cells are harvested in 50-ml aliquots, centrifuged, and washed with 10 ml of 150 mM HEPES, pH 7.0. The cell pellets are finally resuspended in 1.5 ml of the same buffer, and glass beads (0.4 mm diameter, BDH) are added up to the meniscus. The cells are lysed by vortexing for 4 × 30 sec, with intermittent cooling on ice. A further 0.5 ml of 150 mM HEPES, pH 7.0, is added and the suspension is vortexed for 30 sec. After allowing the glass beads to settle, the cell extract is transferred to a clean Eppendorf tube and centrifuged for 5 rain. This separates the soluble protein fractions (supernatant) from the insoluble fractions (pellet). Samples of the soluble
380
RECOMBINANTHEMOGLOBIN
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protein fractions (50-I00/xg total protein) and standard Hb A (200 ng-2 /xg, Sigma) in sample buffer [10 mM Tris-HCl, pH 8.0, 2% (w/v) SDS, 5% (w/v) 2-mercaptoethanol, 1 mM EDTA, 0.01% bromphenol blue] are boiled immediately in a water bath and then resolved on 15% reducing SDS-PAGE gels. We use gels of approximately 15 x 18 cm to ensure good separation of the globin chains. Proteins can be detected by staining the gels with Coomassie blue (Fig. 3). Alternatively, proteins can be electroblotted onto Immobilon P membrane (Millipore, Bedford, MA). Nonspecific binding sites on the membrane are blocked by washing in TBS (20 mM Tris-HC1, pH 7.6, 0.15 M NaCI) containing 0.05% (v/v) Nonidet P-40 and 5% (w/v) milk powder for 45 rain. The membrane is incubated with rabbit antihuman Hb antibody (1 : 1000 dilution; Sigma) in TBS containing 0.05% Nonidet P-40 and 0.2% milk powder for 1 hr, washed for 3 × 5 min in the same buffer, and then incubated with peroxidase-conjugated goat antirabbit antibody (1 : 1000 dilution; Sigma) for 1 hr. After washing for 3 × 5 min, immunoreactive proteins are visualized using the ECL detection system (Amersham), according to the manufacturer's instructions.
Assays of Hemoglobin in Cell Extracts There can be considerable variation in levels of rHb A expression between transformants, probably due to differences in plasmid copy number. It is advisable, therefore, to screen several transformants before choosing one for large-scale growth and subsequent purification of rHb A. Analysis of transformants by SDS-PAGE gels gives some indication of differences in expression levels. We also use a spectrophotometric assay in which rHb A in cell extracts is quantitated from the height of the Soret peak in second-derivative spectra, by comparison with standard Hb A of known concentration. 21 It is important to use samples that have been deoxygenated with sodium dithionite to distinguish rHb A in cell extracts 0tmax 430 nm) from the indigenous yeast cytochrome c (~max 415 nm). Because cell extracts can be quite turbid, we find that clarification using polyethyleneimine (PEI) prior to measuring spectra gives better resuits. Methods
PEI [20/,1 of 5% (v/v) solution in 150 mM HEPES, pH 7.0] is added to l ml of cell extract in an Eppendorf tube and the mixture is centrifuged 2I H. van Urk and D. Coghlan, unpublished results.
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PRODUCTION OF HEMOGLOBIN IN YEAST
381
for 30 sec. The clarified supernatant is removed and 12.5 t~l of sodium dithionite [10% (w/v) solution in 150 mM HEPES, pH 7.0] is added. The absorbance spectrum (290-610 rim) of the sample is measured in the second-derivative mode. Dilutions of standard Hb A, treated in the same way, are used to prepare a standard curve. The rHb A content of cell extracts is calculated as a percentage of total soluble protein, determined using Pierce (Rockford, IL) Coomassie protein assay reagent. Purification of rHb A We use cation-exchange chromatography for purifying rHb A from yeast cell lysates. To prevent the formation of metHb during purification, all chromatography buffers are purged thoroughly with helium to displace dissolved oxygen. In addition CO is bubbled through the lysate for 3-5 rain immediately after cell breakage. The rHb A fractions are maintained in the carbonmonoxy form by repurging with CO at each stage of purification. CarbonmonoxyHb can be converted to the oxy form by flushing the solution on ice with oxygen under bright illumination.
Large-Scale Growth of Yeasts and Preparation of Cell Lysates Yeast transformants can be grown on a large scale in shake flasks (12 x 2-liter flasks each containing I liter of medium). We also grow yeasts by fed batch fermentation using established protocols.Z-~ For purification of rHb A from DBY745 transformants it is important to incorporate protease inhibitors [2 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM EDTA] in the cell lysis buffer to prevent protease damage of rHb A. This is not necessary when using DM477. Methods. A suitable transformant is grown overnight in 200 ml of MM containing 2% (w/v) glucose at 30° with shaking. Growth supplements are added throughout as necessary. The overnight culture is diluted 1 : 100 into 12 x l-liter batches of MM containing 2% (w/v) galactose (for pHb3 transformants) or MM containing 2% (w/v) glucose (for DM477 cotransformants). Cultures are grown at 30° with shaking until in the stationary phase of growth (usually 72 hr). Yeast cells are harvested by centrifugation at 7000 g for 20 rain at 4°. The cells are either used immediately or can be frozen at - 7 0 ° in thin layers in plastic bags for storage at - 20°. The yeast cells are resuspended in 10 mM phosphate buffer, pH 6.5 (containing 2 mM PMSF, 2 mM EDTA if necessary), at a concentration of approximately 60% wet weight/volume 22 S. H. Collins, in "Protein Production by Biotechnology" (T. J, R. Harris, ed.), p. 61. Elsevier, New York, 1990.
382
RECOMBINANTHEMOGLOBIN
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(100 g of cells made up to ~160 ml with buffer). The cells are broken in a Bead Beater (Biospec Products, Bartlesville, OK) using 0.4-ram-diameter glass beads and ice cooling for four 30-sec periods, each separated by a 4-rain interval. The glass beads are removed by vacuum filtration through a glass scinter funnel, and the filtrate is clarified by centrifugation at 30,000 g for 30 rain at 4°. The supernatant is removed, taking care not to disturb the surface lipid layer, and is immediately purged with CO for 5 rain. The extract is kept on ice throughout. The CO-purged lysate is desalted using a Sephadex G-25 (Pharmacia, Piscataway, NJ) column (5 x 30 cm; -~600 ml) equilibrated at 4° with l0 mM phosphate buffer, pH 6.5. The column eluate is monitored at A28o,m and the main protein fraction is collected. The pH of the desalted eluate is adjusted if necessary to pH 6.5 with glacial acetic acid. The conductivity of the eluate should be reduced, if necessary, to less than 2 millisieverts by adding Milli Q water. The eluate is repurged briefly with CO before the next step in purification.
Ion-Exchange Chromatography Desalted eluates are subjected to three successive ion-exchange chromatography steps23: S-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Q Sepharose Fast Flow. Methods. The desalted extract is applied to a 50-ml (3.5 x 5 cm) S-Sepharose Fast Flow column (Pharmacia) equilibrated at 4° with I0 mM phosphate buffer, pH 6.5, at a flow rate of 2.5 ml/min. The rHb A is eluted using a linear pH gradient to pH 8.0 (10 mM phosphate) over 10 column volumes using a Pharmacia FPLC chromatography system. The buffers are purged with helium before use. The elution of rHb A (red fractions) is monitored at A280nmand A405,m. The pooled fractions containing rHb A are purged with CO and the pH is reduced to 6.5 by the addition of glacial acetic acid. The S eluate (pH 6.5) is loaded onto a 10-ml (1.2 x 9 cm) CM-Sepharose Fast Flow column (Pharmacia) at 4 ° equilibrated in 10 mM phosphate, pH 6.5, at a flow rate of 1 ml/min. The same elution procedure (linear pH gradient up to pH 8.0) is used as described here. The rHb A elutes as a single major A405 nm peak, and fractions collected are pooled. A 10-ml Q-Sepharose Fast Flow (FF) column (Pharmacia) (1,5 × 6 cm) is equilibrated in 20 mM Tris-HC1, pH 8.5, at 4° and a flow rate of l ml/min. The pH of the CM eluate is adjusted to 8.5 by the addition of 23 D. Coghlan, unpublished results.
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PRODUCTION OF HEMOGLOBIN IN YEAST
1
2
3
4
383
5
FIG. 4. SDS-PAGE analysis of rHb A purification fractions. Samples are resolved on a 20% homogeneous Phast gel (Pharmacia) and stained with Coomassie blue. All lanes, except lane 2, are loaded with a total of I ~g Hb. Lane 1, standard Hb A (Sigma); lane 2, yeast cell lysate (2 ~g total protein); lane 3, S-Sepharose eluate; lane 4, CM-Sepharose eluate; and lane 5, Q-Sepharose eluate. On these gels a- and/3-globins comigrate.
2 M Tris base and is repurged with CO. This is then loaded onto the Q - S e p h a r o s e FF column at 1 ml/min. The flow rate is reduced to 0.5 ml/ rain or less, and the r H b A is eluted stepwise using either 20 m M bis-Tris buffer, p H 6.6, or 20 m M H E P E S , p H 6.6. This step not only gives an i m p r o v e m e n t in purity but also allows buffer exchange and is an important concentration step.
Purity Assessment The purity of r H b A is monitored during the purification either by s p e c t r o p h o t o m e t r y or by using reducing S D S - P A G E . A rapid quantitative estimation of purity can be obtained f r o m UV/Vis scans (250-650 nm) of r H b A fractions and standard Hb A in their c a r b o n m o n o x y forms, using the following c o m p a r a t i v e a b s o r b a n c e ratios: (A419/Az76)rHb A/(A419/ A276)Hb A. F o r S D S - P A G E , fractions containing r H b A at a concentration of approximately 1 mg/ml in sample buffer (see above) are resolved either on 20% h o m o g e n e o u s Phast gels (Pharmacia) (Fig. 4) or on conventional S D S - P A G E gels.
384
RECOMBINANT HEMOGLOBIN
[24J
Techniques for Physical Characterization of rHb A
Methods HPLC Separation of Globin Chains. A 100-/zl sample of purified rHb A (50-500/~g) is mixed with 100/xl of 8 M urea and chromatographed on an HPLC Vydac C4 reverse-phase (RP) column. 24 The column is equilibrated at 1 ml/min in 70% solvent A:30% solvent B [solvent A is 0.1% trifluoroacetic acid (TFA) in water; solvent B is 90% acetonitrile/0.09% TFA]. Solvent B is increased to 45% over 5 rain, followed by a 30-rain linear gradient to 55% B. Separation of heine,/3-globin, and c~-globin is monitored at A214 nm and A280 nm" Peptide Mapping. Samples (200-500 t~g rHb A or isolated a- or /3-globin chains) in 500/zl of buffer are precipitated in an Eppendorf tube by adding 1 ml of acetone. After vortexing, the tubes are incubated at - 20° for I hr and then centrifuged for 10 rain. The supernatant is discarded; the pellets are dried briefly by rotary evaporation and are then resuspended in 100/A of 6 M guanidine hydrochloride in 0.5 M Tris-HCl, pH 8.0, 2 mM EDTA. The samples are reduced [10/zl of 100 mM dithiothreitol (DTT) in water, 37° for 1-2 hr], carboxyamidomethylated [20 tzl of 100 mM iodoacetamide (Sigma) in 0.1 M Tris-HCl, pH 8.0, 37° for 2-3 hr in the dark], and then diluted 1 : 3 by adding 400/zl of water. Samples are digested with 3 × 5-/.d aliquots of trypsin (Sigma, 1 mg/ml in 1 mM HC1, stored at 4 °) over 48 hr at 37° with shaking. The digests are separated on a Pharmacia Pep-S RP-column. The column is equilibrated in 95% solvent A : 5% solvent B (solvent A is 0.1% TFA in water; solvent B is 70% acetonitrile/ 0.085% TFA) at 0.5 ml/min. The peptides are eluted using a linear gradient up to 60% B over 50 min with detection at A214o~. Amino Acid and N-Terminal Analysis. The a- and/3-globin chains of rHb A and Hb A are separated by RP-HPLC as described above. Aliquots containing 20/zg of each chain are dried separately and resuspended in 20 tzl of 0.1% TFA. Each chain (15 t~g) is subjected to 10 cycles of automated Edman degradation using an Applied Biosystems 477A protein sequencer and to automated acid hydrolysis and amino acid composition analysis using an Applied Biosystems 420 A amino acid analyzer. Mass Spectrometric Analysis. Purified rHb A (I rag) is dialyzed against Milli Q water and analyzed by Electrospray mass spectrometry (ESMS) using a VG Biotech BIO-Q mass spectrometer (M-Scan Ltd., Ascot, UK). 24 S. Rahbar and Y. A s m e r o m ,
Hemoglobin 13, 475 (1989).
[24]
PRODUCTION OF HEMOGLOBIN IN YEAST
385
Functional Studies The simplest and most direct methods to confirm that purified yeastderived rHb A is fully functional rely on optical absorption spectroscopy. We have used such an approach to determine the oxygen equilibrium curves and CO combination kinetics for the recombinant protein. These methods have been described extensively elsewhere. 2-s'26 Only minimal details of the most straightforward techniques will be described here. Prior to functional studies, rHb A purified in the carbonmonoxy form is converted to the oxy form by exposure on ice to a stream of humidified pure oxygen under illumination (Osram Dulux EL.20W low-heat bulb) with periodic agitation for approximately 1 hr. Control experiments are carried out with standard Hb A purified from erythrocytes using established procedures. 27
Oxygen Equilibrium Curves Oxygen equilibrium curves may be obtained conveniently without the requirement for specialized apparatus either by (1) serial addition of air to a modified optical cuvette containing a d e o x y H b solution under vacuum or by (2) controlled enzymatic deoxygenation of a solution of o x y H b in an optical cuvette equipped with an oxygen electrode. Titration Method, The titration method has been described adequately elsewhere. 26 This method is simple to use providing that care is taken to avoid excessive bubble formation and hence denaturation during the evacuation procedures needed to generate d e o x y H b , and that full equilibration is achieved after each addition of air. Equilibration is ensured by transfer of the sample to the tonometer bulb (Fig. 5) by tilting the apparatus, which is then agitated in a water bath for 5 rain. An example of an oxygen equilibrium curve obtained for rHb A using this method is given in Fig. 6. Enzymatic Method. Enzymatic depletion of oxygen has the advantage that Hb is not subjected to lengthy evacuation procedures and also that the cuvette remains at all times within the spectrophotometer housing, thus eliminating repositioning errors. The disadvantages are that the substrates and/or products of the enzymes used to consume oxygen may degrade Hb. We have found that a suitable method uses cytochrome c oxidase zs E. Antonini and M. Brunori, in "Hemoglobin and Myoglobin in Their Reactions with Ligands, Frontiers in Biology" (A. Neuberger and E. L. Tatum, eds.). North-Holland Publ., Amsterdam, 1971. 26 B. Giardina and G. Amiconi, this series, Vol. 76, p. 417. 27A. Riggs, this series, Vol. 76, p. 5.
386
RECOMBINANT HEMOGLOBIN
vacuum pump
[24J
_
4crn
FIG. 5. Diagram of tonometer bulb used to determine oxygen equilibrium curves. The total combined volume (approximately 150 ml) of the tonometer and cuvette must be determined by weighing the system empty and when completely filled with a liquid of known density. A sample of hemoglobin is deoxygenated by evacuation, and the deoxy spectrum is taken, The volume of the sample following deoxygenation is similarly determined by weight. Additions of 0.5-2 ml of air (21% oxygen) at known atmospheric pressure and humidity are made via the vaccine cap as shown. Tonometers may be modified for use with small volumes. 25
and ascorbate/cytochrome c. Alternatively, ascorbate oxidase/ascorbate may be used. These systems reduce oxygen to water as opposed to hydrogen peroxide. It is important to remove the oxygen in about 20 min to minimize the risk of ascorbate damage to the heine group. 28 Methods. A simple system comprises a normal 1-cm pathlength optical cuvette, with ground aperture, into which an oxygen electrode (Yellow Springs Instrument Company, Yellow Springs, OH) can fit tightly. A small gap should remain at one point through which the reactants can be injected using a microsyringe. It is essential that the electrode protrudes into the cuvette and is close (1 cm) to a magnetic follower at the bottom of the cuvette, because the response of the electrode is critically dependent on efficient stirring of the solution close to the membrane. A suitable system 28 j. C. Docherty and S. B. Brown, Biochem. J. 207, 583 (1982).
[24]
387
PRODUCTION OF HEMOGLOBIN IN YEAST
0.3 1.0 0.2 Y 0.5 0,1
0 500
0
550 Wavelength (rim)
600
I
0
~ _ _
10 Oxygen pressure (mm Hg)
I
20
FIG. 6. Determination of o x y g e n equilibrium curves. (a) Spectral transition from deoxyto o x y r H b A (25 ~ M h e m e in 30 m M H E P E S , pH 7.0, 0.2 M NaCI, 25 °) (a --~ k) on titration with air. The final s p e c t r u m was obtained under pure oxygen. The spectra are characteristic of authentic h u m a n Hb A. (b) The fractional saturation (Y) determined from ,£4s76_560,m is plotted against o x y g e n pressure ( m m Hg). The equilibrium curve is characterized by a Ps0 (the partial pressure at which Hb is 50% saturated) of 9.0 m m Hg, nm~~ = 2.9.
comprises Hb at a concentration of 25-100 /xM heine, I0 mM sodium ascorbate, 50/,M tetramethylphenylenediamine (TMPD; electron mediator, Sigma), 0.25/,M cytochrome c (Type VI, Sigma), and 0.5/,M (total heine) bovine cytochrome c oxidase (prepared according to Yonetani 29) in 0.1 M sodium phosphate buffer, pH 6.8, containing 0.05% (v/v) Tween 80, a nonionic detergent to solubilize the enzyme. Alternatively, 2 units ascorbate oxidase (Sigma) can be substituted for cytochrome c oxidase, and the TMPD, cytochrome c, and Tween 80 can be omitted. In both systems the enzyme should be sufficiently active to deoxygenate the solution within about 20 rain. Oxygen equilibrium curves are obtained by plotting the absorbance at a single wavelength (e.g., 576 nm) against the calibrated output from the oxygen electrode. Alternatively, spectra may be recorded throughout oxygen depletion as in Fig. 6. These must, however, be taken at rapid scan rates to ensure minimal distortion due to oxygen depletion during the course of each scan) ° 29 T. Yonetani, J. Biol. Chem. 236, 1680 (1961). 3o K. D. Vandegriff and R, I. Shrager, this series, Vol. 232.
388
RECOMBINANT HEMOGLOBIN
[24]
Carbon Monoxide Binding Kinetics The autocatalytic time course for CO binding to Hb is a convenient probe for the T to R transition and hence functional integrity. The kinetics of binding may be followed at low protein concentration because d e o x y H b tetramers are stable, and dissociation into dimers does not occur in the micromolar concentration range. 3j Methods'. O x y H b (5 to I0 ml of approximately 5/~M heine in 0.1 M phosphate buffer, pH 7.0) solution is partially deoxygenated by three cycles of evacuation and flushing with nitrogen and then drawn into a syringe. Full deoxygenation is achieved by addition of a few grains of solid sodium dithionite or by addition of a 200 mM solution of sodium dithionite made up in anaerobic buffer to a final concentration of 2 raM. This solution is mixed in a standard stopped-flow apparatus (Applied Photophysics, Leatherhead, U K) with solutions of CO in buffer containing 2 mM sodium dithionite. Solutions of CO, at required concentrations (e.g., 25-200 p~M), are prepared by diluting a saturated solution of CO in water (equilibration of water at 20 ° with CO at atmospheric pressure (101 kPa) gives a 1.03 mM solution of CO). Rapid-mixing devices designed to be used with spectrophotometers (e,g., HiTech, Salisbury, UK) are also suitable for use at low CO concentrations. In this case it is often necessary, because of the slow response of the A/D converter, to use an analog signal from the spectrophotometer both for display on an oscilloscope and for conversion to digital form for analysis. Absorbance changes at 436 nm are recorded as a function of time in the l-msec to l-sec time range. At pH 7.0 in 0.1 M phosphate buffer at 20 °, the time course for CO combination to d e o x y H b is autocatalytic, reflecting the T to R transition on CO binding. A series of time courses over a range of CO concentrations allow the second-order rate constant (l') to be determined by standard procedures. 25 The rate constant under these conditions increases from approximately 1 × 10~ M -1 sec J to 2 × 105 M -1 sec -1 throughout the course of binding (see Table I). The time course for CO combination to rHb A should be free of any fast initial component (/' > 106 M -1 s e c - t ) , indicating the presence of free or noncooperative subunits.
Results and Comments Using the purification methods described, we routinely obtain rHb A of >95% purity, with an overall recovery of 50% even when starting with yeast transformants producing rHb A at as little as 0.5% of the total 31 B. W. Turner, D. W. Pettigrew, and G. Ackers, this series, Vol. 76, p. 596.
[24l
389
PRODUCTION OF HEMOGLOBIN IN YEAST
TABLE 1 PHYSICAL AND FUNCTIONAL CHARACTERISTICS OF YEAST-DERIVED STANDARD H b A FROM ERYTHROCYTES
Parameter Spectroscopic analysis
RP-HPLC analysis Peptide mapping N-terminal analysis Amino acid analysis Electrospray mass spectrometry
Oxygen binding properties
CO binding properties
rHb A
AND
Characteristics Normalized spectra of the oxy, deoxy, and carbonmonoxy derivatives of rHb A and Hb A are superimposable between 350 and 650 nm Stoichiometry of c~-globin:/3-globin : heine ratio is identical to that of Hb A Tryptic maps of rHb A and recombinant c~- and/3globin chains are identical to those of Hb A Recombinant c~- and/3-globin chains have correctly processed N termini Amino acid composition of rHb A is identical to standard Hb A Mass: ~-globin, 15,126.0 Da;/3-globin, 15,866.2 Da. These masses are within 1 Da of those of standard c~- and/3-globins Ps0: rHb A, 9.0 mm Hg; Hb A, 8.9 mm Hg (25 tzM heine, 30 mM HEPES, pH 7.0, 0.2 M NaCI, 25°) nn~a~: rHb A, 2.9; Hb A, 3.0 Bohr effect [A log Pso/A pH (pH 7.0 - 7.6)]: rHb A, -0.37; Hb A, -0.34 l ' : r H b A , 2.19 x 0.05 ± l0 sM l s e c - ~ ; H b A , 2.02 x 0.03 +- l05 M -t sec -t (2/xM heine. 0.1 M sodium phosphate, pit 7.0, 20°)
s o l u b l e cell p r o t e i n . R e p r e s e n t a t i v e r e s u l t s for t h e p h y s i c a l a n d f u n c t i o n a l c h a r a c t e r i z a t i o n o f r H b A e m p l o y i n g t h e t e c h n i q u e s o u t l i n e d a r e g i v e n in T a b l e I. P h y s i c a l a n a l y s i s d e m o n s t r a t e s t h a t r H b A is i d e n t i c a l to H b A purified from erythrocytes. The spectroscopic and chromatographic techn i q u e s a l s o c o n f i r m t h a t t h e y e a s t - d e r i v e d p r o t e i n c o n t a i n s a full c o m p l e m e n t o f e n d o g e n o u s l y p r o d u c e d h e m e g r o u p s (i.e., f o u r h e i n e g r o u p s p e r tetramer), In addition, equilibrium and kinetic measurements of the oxygen a n d C O b i n d i n g p r o p e r t i e s o f r H b A d e m o n s t r a t e t h a t the f u n c t i o n a l c h a r a c t e r i s t i c s o f t h e r e c o m b i n a n t p r o t e i n a r e i n d i s t i n g u i s h a b l e f r o m H b A. T h e a b i l i t y to s y n t h e s i z e a u t h e n t i c h u m a n H b A in a s i m p l e e u k a r y o t e , s u c h as S. cerevisiae, p r o v i d e s an i d e a l s y s t e m for t h e e x p r e s s i o n o f engineered mutant Hbs for structural and functional studies. The coexpression systems and purification protocol described here can be easily a d a p t e d for t h e p r o d u c t i o n o f m u t a n t H b s for f u r t h e r a n a l y s i s . M o r e o v e r , genetically engineered yeast producing recombinant human Hb A or mut a n t H b s offers t h e o p p o r t u n i t y f o r p r o v i s i o n o f H b for d e v e l o p m e n t into Hb-based oxygen carriers.
390
RECOMBINANTHEMOGLOBIN
[25]
Acknowledgments We would like to thank many of our colleagues for helpful discussion during the preparation of this manuscript, and especially Bernard Chan, Sarah Gilbert, Katharine Denton, Gareth Jones, David Coghlan, and Henk van Urk for allowing us to refer to their unpublished data. We also thank Kelly Morris for preparing the manuscript.
[25] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f R e c o m b i n a n t H u m a n S i c k l e H e m o g l o b i n E x p r e s s e d in Y e a s t
By
JOSE JAV1ER M A R T I N DE L L A N O , O L A F S C H N E E W I N D , GARY L. STETLER, and JAMES M. MANNING
In the genetic disease sickle cell anemia the abnormal hemoglobin S (Hb S) that is expressed has the hydrophobic side chain of Val-6(/3) in place of the hydrophilic side chain of Glu-6(/3) in hemoglobin A. This substitution on the exterior of the protein causes the aggregation of deoxygenated hemoglobin S tetramers, leading to the distortion of erythrocytes in the venous circulation. I-8 Although the initial aggregation contact is between Val-6(/3) and the Phe-85(/3)/Leu-88(/3) region of an adjacent Hb S tetramer, there are many subsequent points of contact that strengthen the overall stability of the polymer at low oxygen tensions. 3-5 Although the identity of some of the contact sites in the aggregate is known, 3-5'9 there is a lack of information about other contract sites as well as their relative contribution to the overall strength of the aggregate. The number and nature of such susceptible sites have been limited mainly to those hydrophilic side chains with enhanced reactivity because of their location in the protein. 8'~° The availability of recombinant DNA technology now permits studies at any site on the hemoglobin S tetramer, heretofore not possible to modify by other methods. For hemoglobin S, the ability to alter hydrophobic sites 1 L. Pauling, H. Itano, S. J. Singer, and I. C. Wells, Science 110, 543 (1949). 2 V. M. Ingram, Nature (London) 178, 792 (1956). 3 B. C. Wishner, K. B. Ward, E. E. Lattman, and W. E, Love, J. Mol. Biol. 98, 179 (1975). 4 S. J. Edelstein and R. H. Crepeau, J. Mol. Biol. 134, 851 (1979). 5 T. E. Wellems and R. Josephs, J, Mol. Biol. 135, 651 (1979). 6 j, B. Herrick, Arch. Intern. Med. 6, 517 (1910). 7 A. Cerami and J. M. Manning, Proc. Natl. Acad. Sci, U.S.A. 68, 1180 (1971). 8 j. Dean and A. N. Schechter, N. Engl. J. Med. 299, 752, 804, 863 (1978). 9 R. M. Bookchin, R. L. Nagel, and H. M. Ranney, J. Biol. Chem. 242, 248 (1967). t0 j. M. Manning, Adv. Enzymol. Mol, Biol. 64, 55 (1991).
METHODS IN ENZYMOLOGY,VOL. 231
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