Expression and Purification of Recombinant

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Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to form the ferric sulfide complex needed to transport H2S to the bacterial endosymbiont.

Journal of Protein Chemistry, Vol. 20, No. 4, May 2001 (© 2001)

Expression and Purification of Recombinant Hemoglobin I from Lucina pectinata Tanya Rosado-Ruiz,1 Frances M. Antommattei-Pérez,1 Carmen L. Cadilla,2 and Juan López-Garriga1,3 Received April 26, 2001

Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to form the ferric sulfide complex needed to transport H2S to the bacterial endosymbiont. To further study HbI, expression studies of this protein were performed in Escherichia coli. This is the first time that the recombinant HbI was produced using a recombinant DNA expression system. Hemoglobin I cDNA was amplified and cloned into the TOPO-PBAD expression vector, which contains a fusion tag of six histidine residues (6XHis tag). Plasmid clone sequence analysis was carried out in order to ensure that the insert was in the correct reading frame for proper protein expression in E. coli. The expression of recombinant HbI was optimal when induced for 5 hr with 0.002% of L-arabinose as detected by Western blot analysis. The proto-porphyrin group was inserted into the recombinant HbI. Purification of the heme-bound recombinant protein was performed under native conditions by affinity chromatography using Ni-NTA and Probond resins. The sodium dithionite-reduced recombinant protein presented a shift from the Soret band at 413–435 nm, indicating the presence of the heme group in the adequate amino acid environment of HbI. These results indicate that recombinant HbI from Lucina pectinata can be successfully expressed in a prokaryotic system retaining its activity toward reduction, oxidation, and ligand binding. KEY WORDS: Lucina pectinata; recombinant hemoglobin I; H2S transport; recombinant heme protein.

symbiotic bacteria, which need to be supplied with both hydrogen sulfide and oxygen. This mollusk represents a peculiar invertebrate organism characterized by the presence of various cytoplasmic hemoglobins at high concentrations. L. pectinata contains three types of hemoglobins, HbI, HbII, and HbIII (Read, 1962, 1965). The monomeric HbI is a sulfide-reactive protein that binds O2 with comparable affinity to sperm whale myoglobin but is oxidized to the ferric HbI:sulfide complex in the presence of trace oxygen and hydrogen sulfide concentrations (Kraus and Wittenberg, 1990). This property of HbI allows the symbiotic relationship between the clam and the sulfide-oxidizing bacteria. The affinity of HbI for hydrogen sulfide (K ⫽ 2.9 ⫻ 108 M⫺1) is very high in comparison to other hemoglobins. Moreover, in contrast to vertebrate hemoglobins, it carries its functional activity in the ferric state (Kraus and Wittenberg, 1990). It has been postulated that HbI reacts with H2S to form a heme ferric sulfide complex to transport H2S to

1. INTRODUCTION The hemoglobins of vertebrate and invertebrate organisms have been widely studied in order to understand the structure–function relationships, ligand-binding properties, and hemoglobin folding distribution (Urich, 1990). Invertebrate organisms have been shown to have hemoglobins with diverse structure and binding properties (Tokishita et al., 1997; Vinogradov et al., 1993). Lucina pectinata is a bivalve mollusk which inhabits sulfide-rich muddy mangrove areas along the southwest coast of Puerto Rico. This clam houses chemoautotrophic endo-


Chemistry Department, University of Puerto Rico, Mayagüez Campus, Mayagüez, Puerto Rico 00681. 2 Biochemistry Department, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 00936-5067. 3 To whom correspondence should be addressed; e-mail: [email protected]

311 0277-8033/01/0500-0311$19.50/0 © 2001 Plenum Publishing Corporation


Rosado-Ruiz, Antommattei-Pérez, Cadilla, and López-Garriga

the bacterial endosymbiont. The other two hemoglobins (HbII and HbIII) in Lucina’s tissue are oxygen-reactive hemoglobins, which remain oxygenated in the presence of hydrogen sulfide. The X-ray crystal structure of the met-aquo HbI was shown to have a unique structural organization of the heme pocket involving residues Gln64(E7), Phe29(B10), Phe43(CD1), and Phe68(E11) (Rizzi et al., 1994, 1996). This peculiar arrangement of phenylalanyl residues at the distal ligand-binding site is unusual for a hemoglobin and had not been observed before in the globin family. This aromatic environment may be required for the H2S ligand-binding and stability properties of HbI. The X-ray structure for the HbI sulfide derivative (Rizzi et al., 1996) suggested the existence of a hydrogen-bonded complex between the Gln64(E7) and the H2S heme moiety. This result explained the slow dissociation kinetics of the HbISH2 complex. Studies of the amino acid sequence of hemoglobin I from L. pectinata showed that HbI is a monomeric protein of 142 amino acid residues (Kraus and Wittenberg, 1990; Rizzi et al., 1994). The nucleotide sequence of the complementary DNA encoding HbI from L. pectinata and the derivation of the corresponding amino acid sequence of HbI (Antommattei-Perez et al., 1999) confirmed the presence of the Gln64 and aromatic residues surrounding the heme pocket. This aromatic environment, i.e., Phe29 (B10), Phe43 (CD1), and Phe68 (E11), forms an ideal structured aromatic “Phe-cage” unique among globin structures (Bashford et al., 1987; Rizzi et al., 1996; Antommattei-Perez et al., 1999) which may stand as the molecular basis for the very high affinity of HbI for hydrogen sulfide. Resonance Raman studies in the low-frequency region (Silfa et al., 1998; Cerda-Colón et al., 1998; Cerda et al., 1999) showed the ␷Fe–S normal mode at 374 cm⫺1 for the HbI–SH2 complex. Similarly, for the HbIO2, HbICO, and HbICN complexes, the ␷Fe(II)O2, ␷Fe(II)C, and ␷Fe(III)C vibrational modes appear at 563, 516, and 448 cm⫺1, respectively. The vibrational frequencies for the HbI complexes are lower in energy than the same normal modes for other heme proteins. Thus, these results suggested two different HbI heme-ligand amino acid stabilization mechanisms. The first mechanism invokes the presence of H bonding between the heme–Fe(III)SH2 moiety and Glu(64) E7 to stabilize the HbISH2 center. The second mechanism predicts multipolar interactions between the Phe(29)B10 and Phe(68)E11 of the HbICO, HbIO2, and HbICN chromophores to stabilize the CO, O2, and CN⫺ ligand, respectively. Similarly, ferryl heme [Fe(IV)⫽O] species, i.e., compound I and compound II, have been identified as the main intermediates in the reaction of HbI from L. pectinata with hydrogen peroxide (De Jesús-Bonilla et al., 2001). Compound I appears to

be relatively stable, displaying an absorption at 648 nm. The rate constant value for the conversion of compound I to compound II is 3.0 ⫻ 10⫺2 sec⫺1, more than 100 times smaller than that reported for myoglobin. The rate constant for the oxidation of the ferric heme is 2.0 ⫻ 102 M⫺1 sec⫺1. These values suggested an alternate route for the formation of compound II avoiding the step from compound I to compound II. The stabilization of compound I is attributed to the unusual collection of amino acid residues (Gln64, Phe29, Phe43, Phe68) in the heme pocket active site of the HbI protein. Despite all the studies on HbI from L. pectinata there are some structural issues that are not totally clear. Given that the amino acid residues near the heme pocket influence the functional and structural properties of proteins, site-directed mutagenesis is an excellent tool for the study of this protein structure–function relationship. Introducing specific mutations at the amino acid residues (Gln64, Phe29, and Phe68) responsible for the unusual ligand properties of Lucina HbI will help clarify its unresolved structural issues. Protein synthesis through cDNA expression is an important starting point for conducting site-directed mutagenesis studies of HbI from L. pectinata. The optimum expression conditions presented here for the synthesis of a recombinant HbI from L. pectinata demonstrate that recombinant HbI binds the heme group and can be produced successfully in a prokaryotic expression system.

2. MATERIALS AND METHODS 2.1. Synthesis and Cloning of cDNA Lucina pectinata ctenidias were dissected, weighed, and immediately frozen at ⫺70°C in Oak Ridge Tubes. Total RNA was extracted by phenol and guanidinium thiocyanate (Chomczynski and Sacchi, 1987; Antommattei-Perez et al., 1999) using TRI-REAGENT (Molecular Research Center, Inc., Cincinnati, OH). Synthetic oligonucleotides were used as primers for cDNA synthesis by reverse transcriptase–polymerase chain reaction (RT-PCR). The sequence’s were 5⬘-TCTCTCTCTGCTGCACAG-3⬘ for the forward primer and 5⬘-CATGTTTGGTCTGATCAT-3⬘ for the reverse primer. These primers were designed using the HbI cDNA sequence obtained previously (Antommattei-Perez et al., 1999). Synthesis of cDNA was performed with the GeneAmp RNA PCR Kit (Perkin Elmer). The PCR program for amplification of cDNA was as follows: 60 sec at 95°C for denaturation, 35 cycles consisting of 10 sec at 95°C for annealing and 15 sec at 60°C for primer extension, with a final extension time of 7 min at 60°C. The 426-

Recombinant Hemoglobin I from Lucina pectinata bp-long PCR product was cloned in the pBAD TOPO vector (Invitrogen) and transformant clones were isolated (Newman and Fugua, 1999). The presence of the cloned insert was verified by restriction enzyme digestion with NcoI and PmeI (Sambrook et al., 1989). Plasmids containing cloned inserts were sequenced by the dideoxy-chain termination method using dye terminator chemistry (ABI PRISM Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit) in an automated DNA sequencer ABI PRISM 377 (Applied Biosystems, Inc.).

2.2. Hemoglobin I Expression The pTR HbI plasmid, which contained the HbI cDNA insert cloned in the correct reading frame, was used for the protein expression experiments. Recombinant colonies were inoculated into 60 ml of Luria Broth (LB) (10 g/L bacto-tryptone, 5g/L bacto-yeast, and 10 g/L sodium chloride) containing 50 ␮g/ml ampicillin and placed in a shaker incubator at 250 rpm and 37°C for 16 hr. Two 1-L Erlenmeyer flasks containing 500 ml of LB with ampicillin were inoculated with 25 ml of the overnight culture and grown using vigorous shaking (250 rpm) until an A600 reading of 0.5 was reached. At this point, 1-ml cell aliquots were taken and centrifuged at maximum speed for 1 min in a microcentrifuge. The supernatant was aspirated and the pellet was stored at ⫺20°C. Cultures were grown at 37°C in the presence of 0.002% arabinose with shaking for 5 hr. Then, cultures were centrifuged and pellets stored at ⫺20°C. Western blot analysis was used to detect the expressed protein on SDS gels, using a commercial antibody (QIAGEN) against the 6XHis tag of the recombinant protein.

2.3. Heme Group Insertion and Protein Purification under Native Conditions Twenty milliliters of native binding buffer, pH 7.8, was added to the cell pellet obtained followed by addition of egg white lysozyme (final concentration of 1 mg/ ml). The sample was placed on ice for 30 min and then sonicated six times on ice for 30-sec bursts. The insoluble debris was removed from the supernatant by centrifugation of the sample for 30 min at 3000 rpm. The supernatant was transferred to a fresh tube, followed by addition of a 0.15 mg/ml hemin chloride solution to the supernatant in fractions of 1 ml while the sample was in continuous stirring. The total volume of hemin chloride solution added was 10 ml. The sample was stored overnight at 4°C under constant stirring, prior to purification, to allow complete insertion of the heme group into the recombinant HbI protein (Alam et al., 1994).

313 The recombinant HbI protein was purified under native conditions using both the Xpress Purification System™ (QIAGEN) and ProBond™ resin (Invitrogen) (Hoffman and Roeder, 1991). The protein was bound to the resin resuspending a preequilibrated column with three 10-ml native lysate aliquots. The column was rotated for 30 min each time that the protein extract was added to the resin. The resin was settled by low-speed centrifugation. The column was washed three times with 4 ml of the native binding buffer (20 mM phosphate and 500 mM NaCl), pH 7.8, followed by washing with native wash buffer, pH 6.0, until the absorbance at 280 nm was less than 0.01, followed by five additional washes with the native wash buffer, pH 6.0. The protein was eluted from the column in 1-ml fractions by imidazole elution, varying the concentration of imidazole in the elution buffer from 200 to 300 mM. Samples were concentrated in centrifugal filter devices (10,000 MW cutoff; Centricon, Amicon Inc.) for 1 hr and analyzed by SDS–PAGE. To determine if the heme group could be inserted in the heme pocket of the recombinant HbI protein, UV-VIS spectra of the purified protein were obtained (Alam et al., 1994).

3. RESULTS AND DISCUSSION Figure 1 shows that the HbI-coding-region cDNA sequence obtained in this work was the same as the sequence obtained previously (Antommattei-Perez et al., 1999). The cDNA-coding-region sequence contains 426 bp that code for 142 amino acids of HbI, including the Gln64(E7), Phe29(B10), Phe43(CD1), and Phe68 (E11) residues. The alignment with globin sequences (available in the Protein Data Bank and Genebank) shows the highest identity (32%) between Lucina HbI and Chironomus Hb VIIA, and Lucina HbII and HbIII. Similarly, HbI from L. pectinata shares 29%, 29%, 27%, 25%, 23%, and 18% similarity with B. leachii Mb, A. limacina Mb, C. soyoae HbI and HbII, D. auricularia Mb, B. glabrata Mb, and sperm whale Mb, respectively (Antommattei-Perez et al., 1999). Figure 2 indicates that an effective expression of the recombinant protein was obtained after 5 hr of induction with 0.002% of L-arabinose. The expressed protein obtained in this work is a fusion protein, which contains a C-terminal 6XHis tag and a V5 epitope, both used for protein detection and purification. Figure 2b presents the results of the Western blot analysis of the his-tagged protein after 5 hr of induction at various L-arabinose concentrations. Lanes 2 and 15 contain the optimum point of protein expression with L-arabinose. The Western blot indicates a molecular weight for the recombinant protein


Rosado-Ruiz, Antommattei-Pérez, Cadilla, and López-Garriga

Fig. 3. SDS–PAGE of the recombinant protein after the heme group insertion and protein purification. Lane 1, Kaleidescope protein molecular weight standard. Lane 2, Broad-range molecular weight standard. Lanes 3–6, Purified protein eluted with imidazole at 200 mM. Lanes 7–14, Purified protein eluted with imidazole at 300 mM. Fig. 1. Partial nucleotide sequence of the cloned cDNA and its derived amino acid sequence. The underlined regions were used for primer design to perform the synthesis of the cDNA.

of approximately 19.8 kDa. The HbI-calculated molecular weight from the cDNA-derived amino acid sequence is 14,812.8 D. This difference in HbI molecular weight is attributed to additional amino acid residues encoded by the vector that are joined to the HbI protein-coding region in the amino terminal and carboxyl terminal of the protein, which increased the molecular weight of the protein by 5 kDa. Figure 3 shows that the recombinant protein obtained after incubation with the inserted heme group and purified under native conditions by affinity chromato-

Fig. 2. Expressed protein detection analysis. (a) The broad-ranged molecular weight standard. (b) The membrane from the Western blot detection after 5 hr of induction varying the L-arabinose concentration. Lanes 2 and 15 show the optimum point of protein expression after 5 hr of induction with 0.002% L-arabinose.

graphy has an apparent molecular weight of 19.8kDa. Despite the additional amino acids encoded by this vector, the PBAD expression system (Guzman et al., 1995) provides several advantages to control and maximize the protein yield. The TOPO-PBAD expression vector contains the PBAD promoter of the arabinose operon, which offers tight regulation of the expression levels. It also contains the gene encoding AraC, which functions as a positive and negative regulator to provide modulation of the expression levels. These versatile features allowed us to express HbI in a convenient expression system for efficient repression, modulation, and high expression to identify the ideal conditions for its optimal production. Figure 4 shows the UV-VIS data of the purified protein after the heme group insertion with a maximum absorbance at 413 nm. The result suggests the presence of the heme group in the recombinant protein heme pocket. The absorption bands of the recombinant protein occurred at wavelengths close to the absorption band wavelengths of the native HbI from L. pectinata (Silfa et al., 1998; Cerda et al., 1999). The absorbance at 413 nm indicates that the protein obtained after heme group insertion is a combination of both oxy-HbI and metaquo-HbI derivatives, since these species absorb at 416 and 407 nm, respectively. Upon reduction of the recombinant HbI, the band at 413 nm is displaced to 435 nm and visible bands at 530 and 565 nm are also seen. Bands at 433 and 557 nm are observed for the native deoxy-HbI, thus the absorbance bands at 435, 530, and 565 nm for the recombinant protein indicate the presence of the recombinant deoxy-HbI species. Overall, the results suggest that

Recombinant Hemoglobin I from Lucina pectinata


Fig. 4. UV-VIS wavelength scan of the recombinant purified protein after incubation with the hemin chloride solution.

recombinant HbI from Lucina pectinata can be successfully expressed in a prokaryotic system retaining its activity toward reduction, oxidation, and ligand binding. The presence of a known sequenced vector present in the recombinant HbI which increased the molecular weight by 5 kDa can be used as a model to induced protein folding. Current studies also involve site-directed mutagenesis of the HbI cDNA to assess the functionality of specific amino acid residues in H2S binding.

ACKNOWLEDGMENTS This research was supported in part by the National Science Foundation, NSF-MCB9974961 (J.L.G.), the National Institutes of Health, MBRS/SCORE S06GM0810327 (J.L.G.), NCRR-RCMI/G12RR03051 (C.L.C.), and MBRS S06-GM08224 (C.L.C.).

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