Construction of a new polycistronic vector for over-expression and rapid purification of human hemoglobin

Plasmid 61 (2009) 71–77

Contents lists available at ScienceDirect

Plasmid journal homepage: www.elsevier.com/locate/yplas

Construction of a new polycistronic vector for over-expression and rapid purification of human hemoglobin Elisa Domingues, Thomas Brillet, Corinne Vasseur, Virginie Agier 1, Michael C. Marden, Véronique Baudin-Creuza * INSERM U779, University of Paris VII and XI, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France

a r t i c l e

i n f o

Article history: Received 29 July 2008 Revised 16 September 2008 Available online 5 November 2008 Communicated by Richard Calendar Keywords: Co-expression pGEX vector GST PreScission protease Polycistronic system Protein engineering Rapid affinity purification Recombinant Hb A

a b s t r a c t To facilitate the study of the structure-function relationship of human hemoglobin (Hb A), we have developed a new hemoglobin expression vector, pGEX6P-a-[SD]-b. This vector allows the co-expression of a-Hb as a fusion protein with Glutathione S-Transferase (GST-a-Hb) and b-Hb with an additional methionine at the N-terminal extremity (rbHb). These proteins were solubilized as GST-a-Hb/rb-Hb complex form and purified in one step by affinity chromatography on immobilized glutathione. The CO binding kinetic studies show that the GST-a-Hb/rb-Hb complex and recombinant Hb A exhibit the same allosteric behavior as for native Hb A. The GST moiety, which does not modify the function of the complex, can be easily eliminated by cleavage by the PreScission Protease. After cleavage during the rapid purification procedure, over 20 mg of recombinant Hb per liter of culture were obtained, more than double the yield of previous co-expression systems. This polycistronic vector system, which offers the additional advantage of a very rapid purification, is especially well suited for the study of abnormal, unstable globins in order to better understand the associated pathology. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction The normal human adult hemoglobin (Hb A) is a tetrameric protein containing two a-chains and two b-chains, each associated with heme molecule. Different expression systems in bacteria have been developed to produce Hb A in order to better understand the relationship between the structure and the function of Hb and to produce a Hbbased oxygen carrier molecule. The first bacterial system to express the human b- or aglobin was developed by Nagai and Thogersen (1984, 1987). In this system, the globins were produced separately as insoluble cleavage fusion protein. After a laborious purification in denaturing conditions, a semi

* Corresponding author. Fax: +33 1 49 59 56 61. E-mail address: [email protected] (V. Baudin-Creuza). 1 Present address: INSERM U 582, Institut de Myologie, University of Paris VI, 47 boulevard de l0 Hôpital, 75013 Paris, France. 0147-619X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2008.09.006

recombinant Hb was reconstituted in the presence of the native partner subunit and heme with low efficiency. An expression vector was later developed in which the synthetic genes encoding the human a- and b-globin have been produced from a single operon (Hoffman et al., 1990). In this system, the tetramer is produced after incorporation of the exogenous heme as soluble form in the bacterial cytoplasm, and the recombinant human Hb (rHb) contains an additional methionine (Met) residue at the N-terminal of each subunit. The presence of both partner chains and the exogenous heme seems to facilitate the correct folding of the subunits. More recently the authentic soluble human rHb was produced by co-expression both bacterial methionine aminopeptidase (MAP) and synthetic human globin genes under the control of two strong tac promoters (Shen et al., 1993) or with globin genes derived from human globin cDNAs (Shen et al., 1997). In these two expression systems, the purified rHb is obtained at approximately 8 mg/L of culture but requires two steps of purification (Table 1).

72

E. Domingues et al. / Plasmid 61 (2009) 71–77

In 2008, Olson and his group combined mutations in the A helices of subunits and co-expression of bacterial membrane heme transport system in order to enhance apoglobin stability and to increase the rate and extent of the heme capture through the bacterial membranes, respectively (Graves et al., 2008). In certain cases, the expressed yield of this specially mutated rHb in Escherichia coli can be increased over three-fold. However the purification of soluble rHb remains a time-consuming process. An alternative is offered with the versatile pGEX system vector (Smith and Johnson, 1988) that associates the possibility of the high-level expression gene as fusion protein with glutathione S-transferase (GST) to a rapid purification by one step of affinity chromatography. In a first study, we have developed the pGEX4T-AHSP vector which allowed one to express the a-hemoglobin-stabilizing protein (AHSP) at high yield (Baudin-Creuza et al., 2004). The AHSP is described as the chaperone of the a-Hb and forms a stable soluble heterodimer until the association of the a-Hbs with partner b-subunits (Kihm et al., 2002; Gell et al., 2002). In 2006, we showed that the GST-a-Hb subunit alone was produced as an insoluble form with the pGEX4T-a vector. On the contrary, GST-a-Hb subunit was produced as soluble form when it was co-expressed with its chaperone using pGEX4T-a-AHSP vector, AHSP promoting stabilization and solubilization of the a-Hb (VasseurGodbillon et al., 2006a). GST-a-Hb was obtained complexed with its chaperone at high yield after a single affinity chromatography step (Table 1). Addition of b-Hb to the recombinant AHSP/a-Hb complex, with or without GST moiety, leads to the formation of tetrameric Hb with normal allosteric behavior. To remove the GST moiety, this system uses the thrombin protease whose cleavage conditions may lead to the precipitation of the a chains and was not sufficiently adapted to some functional studies of a-Hb. In order to increase the expression of rHb A in E. coli by enhancing the solubility of the expressed proteins particularly for a-subunits and to facilitate the purification, we have developed a pGEX6P-a-[SD]-b vector that allow the co-expression of the a-Hb and the b-Hb under the control of a same tac promoter (Fig. 1). In this objective, only the a-subunits were produced as fusion protein with GST with a PreScission protease cleavage recognition site. Our coexpression system produces the rHb A at high yield by one affinity chromatography step with a-subunits contain-

ing an additional Gly-Pro-Leu-Gly-Ser peptide at the N-terminus (ra-Hb) and b-subunits with only an additional methionine in the N-terminal extremity (rb-Hb). 2. Material and methods 2.1. Plasmids, bacterial strains, and growth media The Hb A expression plasmid pHE7 containing human

a- and b-globin cDNAs was provided by Dr Chien Ho of Carnegie Mellon University (Pittsburgh, PA, USA) (Shen et al., 1997). The expression vector pGEX4T-a containing the human a-globin cDNAs was constructed in the laboratory. The E. coli BL21 (DE3) strains were used for expressing purposes (Lucigen, Middleton, USA). 2.2. Plasmid constructions The pGEX6P-a plasmid was constructed from the pGEX4T-a vector containing the human a-globin cDNA. The pGEX4T-a was digested by BamHI and XhoI and the human a cDNA fragment was inserted between the BamHI and XhoI sites of the pGEX6P-2 vector (GE Healthcare, Lifescience, Uppsala, Sweden). The obtained construction contains a protease cleavage recognition site of the PreScission protease between the GST moiety and a-globin (Fig. 1a). To construct the pGEX6P-a-[SD]-b vector co-expressing GST-a-Hb and rb-Hb, the cassette containing ‘‘Shine and Dalgarno sequence [SD]-human b-globin cDNA sequenceT5ST1T2 terminator region” was amplified from plasmid pHE7 using the two synthetized primers, 50 -CCGCTCGAGCA CCGTGCTGACCTCCAAATACCGTTAAA-30 , and 50 -GCCTGACG TCAGGGTTATTGTCTCATGAGCGGATACATAT-30 , which contain a XhoI site and an AatII site, respectively (Fig. 1b). After restriction enzyme digestion, the PCR product was inserted into pGEX6P-a at the XhoI and AatII sites. The resulting pGEX6P-a-[SD]-b contains the GST-human a-globin cDNA and human b-globin cDNA sequences under the same tac promoter (Fig. 1c). 2.3. Co-expression and solubilization of the GST-a-Hb and rbHb proteins The GST-a-Hb and rb-Hb were expressed in E. coli BL21 (DE3) cells using the co-expression plasmid pGEX6P-a[SD]-b after induction by 0.2 mM isopropyl b-thiogalacto-

Table 1 Comparison of different used plasmids to express soluble human a-subunits, b-subunits or rHb in E. coli Plasmids

Characteristics

Soluble expressed Purification chromatography proteins

Yield (mg/L)

Source

pHE7

Human b- and a-globin cDNAs and E. coli MAP Human b-globin cDNA and E. coli MAP Human a-globin cDNA and E. coli MAP Human a-globin and AHSP cDNAs Human a- and b-globin cDNAs

Hb A and MAP

8–10 mg Hb A 3–4 mg bHb A 3 mg a-Hb A 12–20 mg a-Hb A 16–23 mg Hb A

Shen et al., 1997

pHE2b pHE2a pGEX4T-a-AHSP pGEX6P-a-[SD]-b *

b-Hb and MAP

a-Hb and MAP GST-a-Hb/ GSTAHSP* GST-a-Hb/rb-Hb*

These proteins were obtained in the complex form.

2 Ion-exchange steps: Q-Sepharose fastflow and Mono S 2 Anion-exchange steps: DEAE-cellulose and Mono Q 2 Ion-exchange steps: CM-52 and Source 15S One affinity step with concomitant thrombin cleavage One affinity step with concomitant PreScission protease cleavage

Yamaguchi et al., 1996 Adachi et al., 2000 Vasseur-Godbillon et al., 2006a In this study

73

E. Domingues et al. / Plasmid 61 (2009) 71–77

pTac GST GST α -globin

PP BamHI

α -globin

pGEX6P- α

XhoI

5392 bps

AatII Amp

pBR322 ori

AatII BamHI

1.a

T5S-T1-T2 XhoI

EcoRI pTac

BamHI

HindIII MAP

Amp

β-globin SD β -globin α-globin PP GST

pGEX6P- α -[SD]- β 6119 bps

pBR322 ori

GST α -globin

T5S-T1-T2

pHE7

pTac SD

6824 bps

α -globin SD

pTac

EcoRI Xho I HindIII

β-globin pBR322 ori T5S-T1-T2 Amp

Aat II

SD β -globin T5S-T1-T2

1.c

BamHI EcoRI

1.b Fig. 1. Construction of expression plasmid for human adult Hb. (a) pGEX6P-a contains the gene expression for GST-human a-globin under the control of a tac promoter. (b) From pHE7 vector, the cassette containing Shine and Dalgarno sequence [SD]-human b globin cDNA sequence-T5ST1T2 terminator region was obtained. (c) The resulting pGEX6P-a-[SD]-b contains under the same tac promoter the GST-human a-globin cDNA and human b-globin cDNA sequences.

pyranoside at 37 °C and then supplemented with hemin (30 lg/mL). The growth was continued for 5 h at 37 °C. The bacterial cells were harvested by centrifugation. The cell pellets were suspended in PBS (150 mM NaCl, 10 mM Na2HPO4, pH 7.4) and stored at 80 °C. The GST-a-Hb and rb-Hb proteins were solubilized as previously described (Baudin-Creuza et al., 2004). Briefly, the suspended cells were incubated at 4 °C for 40 min in the presence of lysozyme (1 mg/mL of mixture) and DTT (5 mM). The mixture was sonicated (Branson Ultrasonic, Carouge-Geneva, Switzerland), saturated with CO gas and incubated with Triton X-100 to a final concentration of 1% for 1 h at 4 °C with slight agitation. Then the lysate was clarified by centrifugation at 15,300g at 4 °C for 30 min. 2.4. Purification of the GST-a-Hb/rb-Hb complex and rHb A After centrifugation, the soluble fraction containing the GST-a-Hb/rb-Hb complex was purified in one step by affinity chromatography on Glutathione Sepharose 4B column (GSTrap 4B column, GE Healthcare, Lifescience). After washing with the PBS, the GST-a-Hb/rb-Hb was eluted by glutathione buffer (10 mM reduced glutathione in Tris– HCl 50 mM buffer at pH 8.0). Then the GST-a-Hb/rb-Hb was concentrated by ultrafiltration (Amicon Ultra-15, Millipore, Billerica, MA, USA) and again saturated with CO gas. To obtain the rHb A, the cleavage of the GST moiety was directly performed on the GST-a-Hb/rb-Hb complexes

bound to Glutathione Sepharose 4B column by the addition of PreScission protease (2 units/100 lg of fusion protein) (GE Healthcare, Lifescience) in PBS containing 1 mM DTT and saturated with CO gas. After an overnight incubation at 4 °C, the column was washed with the PBS and the rHb A was recovered in the flow-through while the PreScission protease itself a GST fusion protein remained bound to the resin. 2.5. Characterization of GST-a-Hb/rb-Hb complex and rHb A Different aliquots were collected during expression, solubilization and after purification of recombinant proteins and prepared by boiling in SDS loading buffer. These different samples were analyzed by SDS–PAGE according to Laemmli (1970) and revealed by Coomassie Brilliant Blue staining. After purification, the GST-a-Hb/rb-Hb complex was analyzed by size-exclusion chromatography using a SuperoseTM12 30/100GL (GE Healthcare, Lifescience) eluted at 25 °C with PBS (Baudin-Creuza et al., 2004). After cleavage by PreScission protease, the rHb A was equilibrated in 50 mM Bis–Tris buffer at pH 7.0 by passing through on PD-10 Desalting column (GE Healthcare, Lifescience) and again saturated with CO gas. Then, the rHb A was analyzed by size-exclusion chromatography before and after concentration by ultrafiltration (Amicon Ultra-15, Millipore).

74

E. Domingues et al. / Plasmid 61 (2009) 71–77

UV and visible spectra measurements were carried out with a Varian Cary 400 spectrophotometer. The spectra of the CO form of purified GST-a-Hb/rb-Hb complex and rHb A (5–10 lM on a heme basis) were measured in 50 mM Bis–Tris buffer at pH 7.0. 2.6. Kinetics of CO recombination

3. Results and discussion To enhance the production of the rHb A, we developed a new polycistronic expression vector derived from pGEX6P2 in which the rb-Hb and GST-a-Hb fusion protein were produced in the bacterial cytoplasm (Fig. 1c). In a previous study, we have used the pGEX-4T system for the co-expression of a-Hb with AHSP as GST fusion proteins with a thrombin cleavage recognition site (VasseurGodbillon et al., 2006a). The thrombin has an activity at room temperature and its elimination is more delicate. Depending on experiments, the efficiency of cleavage of the GST-a-Hb/AHSP-GST complex was variable. Hakes and Dixon have reported an improvement of the thrombin cleavage efficiency by the insertion a glycine-rich ‘‘kinker” placed N-terminal to the recognition site (Hakes and Dixon, 1992). Moreover, during the thrombin cleavage of the complex, we have observed a certain level of desheminization of the ra-Hb fraction. In addition, an inhibition of the cleavage was observed when the sample was prepared in the CO form which generally stabilizes the Hb molecules. These problems have not been observed with the pGEX6P system. Indeed the pGEX-6P type vector contains a PreScission protease cleavage recognition site. This protease has two major advantages. It is itself a GST fusion protein that facilitates its elimination and reduces the preparation time of recombinant protein without the GST moiety. The second advantage is its high activity at low temperature. 3.1. Production of the GST-a-Hb/rb-Hb complex and rHb A

Fig. 2. SDS–PAGE (5–24%) analysis of the co-expression of GST-a-Hb and rb-Hb. The patterns showed the different proteins during the expression, the solubilization and the different purification steps. (Lane 1) molecular weight markers (kDa shown on the left); (lane 2) Whole cell lysate after 5 h induction with IPTG; (lane 3) soluble fraction after cell disruption; (lane 4) GST-a-Hb/rb-Hb eluted from Glutathione Sepharose 4B with elution buffer (reduced glutathione 10 mM in Tris–HCl 50 mM, at pH 8.0); (lane 5) rHb A released from Glutathione Sepharose 4B after PreScission protein cleavage; (lane 6) a-Hb control; (lane 7) b-Hb control; (lane 8) mix of a-Hb and b-Hb control.

only 3 arginines, 9 histidines and 11 aromatic residues, which explains the lower sensitivity for the low molecular weight proteins. After solubilization, several bands were found in the soluble fraction, one corresponding to GSTa-Hb (Fig. 2, lane 3). After purification by affinity chromatography on Glutathione Sepharose 4B, the SDS–PAGE study revealed the presence of two major bands corresponding to GST-a-Hb and rb-Hb, respectively (Fig. 2, lane 4). After cleavage of the GST-a-Hb/rb-Hb complex by PreScission protease, the SDS–PAGE analysis showed the presence of only one wide band corresponding to both ra-Hb and rb-Hb (Fig. 2, lane 5) as observed for the mix of a and b Hb controls (Fig. 2, lane 8). 3.2. Spectral and chromatographic characterization of GST-aHb/rb-Hb complex and rHb A The spectra of the ferrous GST-a-Hb/rb-Hb complex and rHb A in the CO form are shown in Fig. 3. The Soret absorption at 419 nm and the amplitude of the a and b absor-

1.0

0.8

Absorbance

The bimolecular recombination kinetics were measured after flash-photolysis using 10 ns YAG laser pulses (Quantel, Les Ulis, France) at 532 nm. Samples at 5–10 lM on a heme basis were in 4  10 mm quartz cuvettes with observation at 436 nm. Measurements were at 25 °C in 50 mM Bis–Tris buffer at pH 7.0, 100 lM CO (Marden et al., 1988). In a second measurement, IHP, an analogue of the 2,3-DPG effector, was added at a final concentration of 1 mM to enhance the fraction of T state of tetrameric Hb. In a third measurement, the laser energy was reduced (to vary the level of the heme photo-dissociation from 50% to 7%) to study the allosteric behavior of the recombinant proteins.

0.6

GST- α -Hb/r β-Hb

rHbA

0.4

HbA

The SDS–PAGE analysis showed that after 5 h induction, the whole cell lysate exhibits one intense band at 40 kDa corresponding to GST-a-Hb (42.57 kDa) and a very weak band at slightly less than 15 kDa corresponding to rb-Hb (16.62 kDa) (Fig. 2, lane 2). The Coomassie Blue stains the protein by binding via the arginine, histidine and aromatic residues. Contrary to GST-a-Hb that has 12 arginines, 16 histidines and 39 aromatic residues, the rb-Hb contains

0.2

0.0 250

300

350

400

450

Wavelength (nm) Fig. 3. Absorption spectra of Hb A, rHb A and GST-a-Hb/rb-Hb for CO bound form. The spectra were measured at 25 °C in 50 mM Bis–Tris buffer at pH 7.0.

75

E. Domingues et al. / Plasmid 61 (2009) 71–77

bance peaks (540 and 571 nm, respectively, data not shown) were identical for the GST-a-Hb/rb-Hb complex and rHb A to that observed for native Hb A. The absorption ratio 419/280 was lowered, from 4.63 for the native Hb A to 2.56 for the GST-a-Hb/rb-Hb, due to the GST moiety. This absorption ratio for rHb A was 4.08 indicating that the subunits of rHb A were correctly heminized. From these ratios and the total absorption, one can determine the quantity of the purified rHb A, which varied from 16 to 23 mg from 1 L of culture (Table 1). Fig. 4 illustrates both purified GST-a-Hb/rb-Hb and rHb A profiles on SuperoseTM12 30/100GL at 280 nm. The same profiles were obtained at 415 nm, specific of the heme protein (profile not shown). The GST-a-Hb/rb-Hb was eluted as two species before the DCL-Hb (diaspirin cross-linked Hb), the undissociated tetrameric Hb control. Based on a calibration curve using globins, the elution volume of the major peak corresponds to 172.8 kDa. Considering the theoretical masses of the GST-a-Hb/rb-Hb (59.2 kDa), this experimental value is consistent with GST-a-Hb/rb-Hbforming trimers (2.92 subunits). Note however that GST is expected to form dimers and estimated MW based on globin calibration may not be valid for the non-globular geometry of the GST complex. This feature has already been reported in previous work where we have shown the presence of molecular size higher when the GST moiety is present (Baudin-Creuza et al., 2004). For rHb A, we observed the presence of a single species which eluted between the reference DCL-Hb and the Hb Rothschild (dimeric control). For concentrations of 79 and 640 lM on a heme basis, the elution volume of this molecular species decreased from 13.59 to 13.34 mL (Fig. 4). Based on a calibration curve, these values correspond to 43.2 kDa and 49.6 kDa, respectively. Considering that the theoretical masses of the ra-Hb and rb-Hb, the recombi-

α2 β2

αβ

α-Hb

3.3. Kinetic studies Hb A exists in two affinity states, a low-affinity state having the quaternary structure of deoxygenated Hb, called T, and a high-affinity state with the quaternary structure of fully liganded Hb, called R. For native Hb A at 25 °C in aqueous buffer, about 50% of CO rebinding is attributed to the nanosecond geminate recombination and 50% to the millisecond bimolecular recombination. The CO recombination kinetics of native Hb A are dependent on the laser energy, since a higher fraction of heme photodissociated will produce more of the deoxy and singly liganded tetramers which switch readily from the R quaternary structure to the T quaternary structure, reflecting the allosteric transition. IHP (inositol hexaphosphate) enhances the observed amount of slow phase (T-like) since this effector strongly favors the T-state even for the doubly liganded tetramers. The percent of slow phase obtained from the CO recombination kinetics after photo-dissociation are shown for native Hb A, rHb A and GST-a-Hb/rb-Hb complex in Fig. 5. The CO rebinding kinetic for the rHb A was similar to that described for the native Hb A exhibiting mainly a T-phase, 10.5% and 13%, respectively. For the GST-a-Hb/ rb-Hb complex, the CO rebinding kinetic showed slightly less of the slow phase (4%) relative to the native Hb A. The calculated values for the rates of CO rebinding for the three samples were identical with 6 lM1 s1 for the rapid phase and 0.4 lM1 s1 for the slow phase. Addition of the allosteric effector IHP leads to an increase of the slow (T-state) kinetic phase. The T-state fraction increased from 10.5% to 50% for rHb A and from 3.5% to 43% for GST-a-Hb/rb-Hb complex. In the case of native

0.8

80

HbA

0.6

rHbA

Slow phase %

Normalized absorbance (280 nm)

1.0

nant ab dimer is 32.77 kDa, the observed values are consistent with those expected for a non-complete transition from dimer [ra-Hb/rb-Hb] at low concentration to tetramer [ra-Hb/rb-Hb]2 at high concentration.

0.4

0.2

0.0

60

GST- α -Hb/r β -Hb

40

20

-0.2 10

12

14

16

Elution volume (mL) Fig. 4. Size-exclusion chromatography profiles on Superose 12 10/300GL column of purified GST-a-Hb/rb-Hb and rHb A. These profiles were obtained in separate chromatographic runs for GST-a-Hb/rb-Hb after purification on Glutathione Sepharose 4B (––) and for rHb A after cleavage on Glutathione Sepharose 4B at 79 lM (  ), and after concentration by ultrafiltration at 640 lM (– –). DCL-Hb (diaspirin cross-linked Hb), Hb Rothschild [b37Trp? Arg] and a-Hb were used as controls for undissociated tetrameric a2b2 Hb, ab dimeric Hb and monomeric Hb, respectively. The absorbance was followed at 280 nm. The experimental conditions were in PBS at 25 °C with a 0.4 mL/min flow-rate.

0

- IHP 50%

+IHP 50%

+IHP 20.2%

+ IHP 7.2%

Fig. 5. Percent of slow phase (T-state) obtained from the CO recombination kinetics after photo-dissociation for Hb A (black), rHb A (gray) and GST-a-Hb/rb-Hb (white). In general Hb tetramers display two kinetic phases, with the slower phase characteristic of the T-state conformation. The kinetics under different conditions can demonstrate the correct allosteric response for Hb. As expected, IHP increases the fraction of slow (T-state) kinetics, while decreasing the fraction dissociation (lower laser energy from 50% to 7.2%) will lead to less T-state. Experimental conditions were 50 mM Bis–Tris buffer at pH 7.0, 100 lM CO at 25 °C in absence or in presence of IHP at a final concentration of 1 mM.

76

E. Domingues et al. / Plasmid 61 (2009) 71–77

Hb A, the T-state fraction went from 13% to 57%. Typical of recombinant systems, rHb A and the GST-a-Hb/rb-Hb complex, thus show a slightly smaller variation and maximum amount of the slow phase. Another test of the allosteric transition is to vary the fraction of ligands bound. At sufficiently low laser energies, the main photoproduct is triply liganded tetramers, which tends to display R-like (rapid) kinetics. The decrease of the photo-dissociated fraction of native Hb A (50% to 7%, heme photo-dissociation at 10 ls) lead to decrease the fraction of slow T-state kinetics (from 57% to 12%). This is expected for the Hb tetramer, where more T-state form occurs when a higher fraction of ligands are photodissociated. The CO bimolecular recombination kinetics of rHb A and GST-aHb/rb-Hb complex were similar to those observed for native Hb A, indicating a normal allosteric behaviour. In all experimental conditions, the purified GST-a-Hb/ rb-Hb complex exhibits CO recombination kinetics after photo-dissociation similar to those observed for Hb A, with a proportion of the T-state which is slightly decreased. This indicates that the GST moiety does not interfere in the association between a and b subunits to form functional a2b2 tetramers. This is also compatible with the expected dimerisation of GST, and not the formation of trimers which would imply an addition ab dimer which are known to be non-allosteric. The functional results show that the rHb A has the same behavior as native Hb A, characterized by the two allosteric states of Hb tetramer. This is consistent with the results of gel filtration that have shown that the rHb A is tetrameric at high concentration. The b1 N-terminus is known to be a binding site of organic phosphates such as IHP and 2,3-DPG (Arnone, 1972; Arnone and Perutz, 1974). In 1990, an expression system was developed to produce fully functional tetrameric human Hb in E. coli, in which each globin contains an additional N-terminal methionine (Hoffman et al; 1990). The oxygen affinity of this recombinant Hb is similar to that of native Hb A. However, reduced Bohr and phosphate effects are observed which may be attributed to the presence of methionine at the N-extremity of the subunits. In other study, Doyle et al. (1992) have shown that in the absence of organic phosphates, the functional behaviour of the two recombinant Hb (b 1 Val? Met), and (b 1 Val + Met) is similar to that of native Hb A. However, the effects of organic phosphates on CO binding kinetic properties of the recombinant Hb (beta 1 + Met) was found to deviate somewhat from normalcy. In our expression system, the CO recombination kinetics show that the presence of an additional methionine in the b Nextremity of the rHb A does not appear to affect its allosteric function in presence of the IHP. In conclusion, we have described an expression system to produce a high yield of rHb A with only one step purification. Our system has a shorter purification time and can increase at least by a factor of two the production yield (Table 1) relative to co-expression systems described by Shen et al. (1997). Unlike the pGEX 4T-a expression vector that produces the ra-Hb subunit in an insoluble form, the new co-expression vector allows production of soluble ra-Hb associated with the partner rb-Hb. These results confirm the stabilizing role of the b-subunits towards a-

subunit and are consistent with those of Adachi and collaborators (2002) suggesting that the a and b subunits associate during or soon after translation acting as folding catalysts to promote tetrameric Hb A formation. This new polycistronic expression vector is a very efficient tool to rapidly produce rHb A or GST-a-Hb/rb-Hb at high yield. Furthermore, the expression system described here may be generally useful for reproducing abnormal chain involved in certain hemoglobinopathies. Indeed we have already shown with the co-expression pGEX4T-aAHSP vector that some unstable ra-Hbs had an impaired interaction with AHSP (Vasseur-Godbillon et al., 2006b). It would be interesting to follow the interaction of these mutants with their partner b-subunits in our new coexpression system. Acknowledgments We thank G. Caron for skilful technical assistance. We are grateful to the Baxter Healthcare Company for supplying DCL-Hb. This work was supported by the Institut National de la Santé et de la Recherche Médicale, the University Paris XI. T. Brillet was supported by the Délégation Générale pour l0 Armement (France), scientist responsible: C. Dane. References Adachi, K., Yamaguchi, T., Yang, Y., Konitzer, P.T., Pang, J., Reddy, K.S., Ivanova, M., Ferrone, F., Surrey, S., 2000. Expression of functional soluble human alpha-globin chains of hemoglobin in bacteria. Prot. Expr. Purif. 20, 37–44. Adachi, K., Zhao, Y., Surrey, S., 2002. Assembly of human hemoglobin (Hb) beta- and gamma-globin chains expressed in a cell-free system with alpha-globin chains to form Hb A and Hb F. J. Biol. Chem. 277, 13415– 13420. Arnone, A., 1972. X-ray diffraction study of binding of 2, 3diphosphoglycerate to human deoxyhaemoglobin. Nature 237, 146–149. Arnone, A., Perutz, M.F., 1974. Structure of inositol hexaphosphate human deoxyhaemoglobin complex. Nature 249, 4–6. Baudin-Creuza, V., Vasseur-Godbillon, C., Pato, C., Préhu, C., Wajcman, H., Marden, M.C., 2004. Transfer of human alpha to beta-hemoglobin via its chaperon protein: evidence for a new state. J. Biol. Chem. 279, 36530–36533. Doyle, M.L., Lew, G., De Young, A., Kwiatkowski, L., Wierzba, A., Noble, R.W., Ackers, G.K., 1992. Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins. Biochemistry 31, 8629–8639. Gell, D., Kong, Y., Eaton, S.A., Weiss, M.J., Mackay, J.P., 2002. Biophysical characterization of the alpha-globin binding protein alphahemoglobin stabilizing protein. J. Biol. Chem. 277, 40602–40609. Graves, P.E., Henderson, D.P., Horstman, M.J., Solomon, B.J., Olson, J.S., 2008. Enhancing stability and expression of recombinant human hemoglobin in E. coli: progress in the development of a recombinant HBOC source. Biochim. Biophys. Acta. 1784, 1471–1479. Hakes, D.J., Dixon, J.E., 1992. New vectors for high level expression of recombinant proteins in bacteria. Anal. Biochem. 202, 293–298. Kihm, A.J., Kong, Y., Hong, W., Russel, J.E., Rouda, S., Adachi, K., Simon, M.C., Blobel, G.A., Weiss, M.J., 2002. An abundant erythroid protein that stabilizes free a-haemoglobin. Nature 417, 758–763. Hoffman, S.J., Looker, D.L., Roehrich, J.M., Cozart, P.E., Durfee, S.L., Tedesco, J.L., Stetler, G.L., 1990. Expression of fully functional tetrameric human hemoglobin in Escherichia coli. Proc. Natl. Acad. Sci. USA 87, 8521–8525. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Marden, M.C., Kister, J., Bohn, B., Poyart, C., 1988. T-state hemoglobin with four ligands bound. Biochemistry 27, 1659–1664. Nagai, K., Thogersen, H.C., 1984. Generation of b-globin by sequencespecific proteolysis of a hybrid protein produced in Escherichia coli. Nature 309, 810–812.

E. Domingues et al. / Plasmid 61 (2009) 71–77 Nagai, K., Thogersen, H.C., 1987. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 153, 461–481. Shen, T.J., Ho, N.T., Simplaceanu, V., Zou, M., Green, B.N., Tam, M.F., Ho, C., 1993. Production of unmodified human adult hemoglobin in Escherichia coli. Proc. Natl. Acad. Sci. USA 90, 8108–8112. Shen, T.J., Ho, N.T., Zou, M., Sun, D.P., Cottam, P.F., Simplaceanu, V., Tam, M.F., Bell, D.A., Ho, C., 1997. Production of human normal adult and fetal hemoglobins in Escherichia coli. Protein Eng. 10, 1085–1097. Smith, D.B., Johnson, K.S., 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31–40.

77

Vasseur-Godbillon, C., Hamdane, D., Marden, M.C., Baudin-Creuza, V., 2006a. High-yield expression in Escherichia coli of soluble human alpha-hemoglobin complexed with its molecular chaperone. Protein Eng. Des. Sel. 19, 91–97. Vasseur-Godbillon, C., Marden, M.C., Giordano, P., Wajcman, H., BaudinCreuza, V., 2006b. Impaired binding of AHSP to alpha chain variants: Hb Groene Hart illustrates a mechanism leading to unstable hemoglobins with alpha thalassemic like syndrome. Blood Cells Mol. Dis. 37, 173–179. Yamaguchi, T., Pang, J., Reddy, K.S., Witkowska, H.E., Surrey, S., Adachi, K., 1996. Expression of soluble human beta-globin chains in bacteria and assembly in vitro with alpha-globin chains. J. Biol. Chem. 271, 26677– 26683.