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Applied and Environmental Microbiology, September 2008, p. 5854-5856, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01291-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System{triangledown}

D. M. Villarreal,1 C. L. Phillips,1 A. M. Kelley,1 S. Villarreal,1 A. Villaloboz,1 P. Hernandez,1 J. S. Olson,2 and D. P. Henderson1*

Department of Biology, University of Texas of the Permian Basin, Odessa, Texas 79762,1 Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 770052

Received 10 June 2008/ Accepted 20 July 2008


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ABSTRACT
 
To produce recombinant hemoglobin in Escherichia coli, sufficient intracellular heme must be present, or the protein folds improperly and is degraded. In this study, coexpression of human hemoglobin genes and Plesiomonas shigelloides heme transport genes enhanced recombinant hemoglobin production in E. coli BL21(DE3) grown in medium containing heme.


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INTRODUCTION
 
Numerous attempts have been made to find an alternative to human blood for patients who require blood transfusions (for recent reviews, see references 1 and 6). One approach is to harvest recombinant hemoglobin expressed in Escherichia coli. When human {alpha}- and β-globin chains are expressed in E. coli, hemoglobin can be recovered from soluble fractions of cells (5). However, the chains are degraded unless sufficient heme is present to allow assembly into tetrameric hemoglobin (13-15). The problem has been addressed previously by adding large quantities of heme to the culture medium of E. coli strains that are more permeable to heme than other strains. Although E. coli can synthesize heme, nonpathogenic strains lack high-affinity heme transport systems.

Plesiomonas shigelloides is an intestinal pathogen that uses heme iron (2). Its heme transport system consists of HugA, the outer membrane heme receptor; TonB and ExbBD, which allow HugA to move heme into the periplasm; HugB, which moves heme across the periplasm; and HugC and HugD, which move heme into the cytoplasm (4). Three additional proteins, HugWXZ, may be required for the utilization of heme as an iron source but not for heme transport (4, 8).

A recent study demonstrated that the expression of a heme receptor gene in E. coli increases the heme content and activity of plasmid-encoded catalase (11). In our study, we assessed hemoglobin production in E. coli BL21(DE3) coexpressing human hemoglobin and P. shigelloides heme transport genes.


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The P. shigelloides heme utilization system supports the growth of BL21(DE3) in heme-containing medium.
 
Heme utilization assays were performed to demonstrate that BL21(DE3) coexpressing human hemoglobin genes (on pHB0.0) and P. shigelloides heme utilization genes (on pHUG17.1) can transport heme and use it as an iron source. BL21(DE3)/pHB0.0 with pHUG17.1 or the parent vector (pWSK29) were grown for 20 h in M9 medium (10) containing 7.6 µM heme (Sigma Chemicals) and 100 µg/ml of the iron chelator ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDA) (Complete Green Company) (9). BL21(DE3)/pHB0.0 with the heme utilization system reached an absorbance of 1.03 at a wavelength of 600 nm, compared to 0.09 for BL21(DE3)/pHB0.0 with the parent vector. Both strains had absorbances of approximately 1.0 in M9 medium with 20 µM FeSO4 and below 0.10 in M9 medium with EDDA and no heme.


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BL21(DE3) containing hemoglobin genes and P. shigelloides heme transport genes produces high levels of hemoglobin.
 
To assess hemoglobin production, BL21(DE3)/pHB0.0 with or without the heme transport system was grown in L broth with 6.25 µg/ml EDDA to allow the expression of heme transport genes. After 8 h, 40 µg/ml isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce the expression of hemoglobin genes which are controlled by a tac promoter, and 15.2 µM heme was added to increase intracellular heme. After 16 h of additional growth, cultures were flushed with carbon monoxide (CO) for 20 min, adjusted to an absorbance of 0.50 at 700 nm, and scanned on a Spectronic Genesys spectrophotometer at 350- to 700-nm wavelengths.

Both cultures showed a peak at 419 nm, consistent with CO hemoglobin (7) (Fig. 1A), but the peak for BL21(DE3) coexpressing the hemoglobin genes (on pHB0.0) and heme transport genes (on pHUG21) was markedly higher than that for BL21(DE3) expressing the hemoglobin genes alone. No peak was detected in the scan of E. coli containing the heme transport genes alone [BL21(DE3)/pDSV1, pHUG21], indicating that the heme transport system did not cause the accumulation of heme-containing proteins which could contribute to the spectrum.


Figure 1
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  		FIG. 1. Hemoglobin production in BL21(DE3) transformed with various plasmids. (A) Absorbance scans of live cultures of BL21(DE3) with and without hemoglobin or P. shigelloides heme transport genes. Solid line, E. coli containing hemoglobin and heme transport genes [(BL21(DE3)/pHB0.0, pHUG21]; dotted line, E. coli containing hemoglobin genes alone [BL21(DE3)/pHB0.0, pWSK29]; dashed line, E. coli containing heme transport genes alone [BL21(DE3)/pDSV1, pHUG21]. Values on the x axis are wavelengths in nanometers. (B) First derivative spectrum obtained from absorbance scans. Derivative values are shown beside each arrow. Solid line, BL21(DE3) containing heme transport and hemoglobin genes; dotted line, BL21(DE3) containing hemoglobin genes alone. (C) Immunoblot of soluble (S) and insoluble (I) fractions from 0.004 OD unit of cells at a wavelength of 700 nm. Lanes 1 and 4, BL21(DE3) containing heme transport genes alone; lanes 2 and 5, BL21(DE3) containing hemoglobin genes alone; lanes 3 and 6, BL21(DE3) containing hemoglobin and heme transport genes; lane 7, 0.45 µg of hemoglobin (Sigma Chemicals).

The first derivative profiles of the absorbance scans were obtained (Fig. 1B). Graves et al. (3) found that first derivative values of live cultures correlate with quantities of purified hemoglobin isolated from cells. When three independent assays were performed, BL21(DE3) with hemoglobin and heme transport genes or with hemoglobin genes alone had average derivative values of 0.134 ± 0.002 and 0.023 ± 0.002, respectively.

To determine if derivative values correlated with levels of soluble hemoglobin, cells were lysed and centrifuged to separate soluble fractions found in supernatants from insoluble fractions found in pellets (13). Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system. BL21(DE3) with hemoglobin and heme transport genes produced substantially higher levels of soluble hemoglobin than with hemoglobin genes alone (Fig. 1C, lanes 3 and 2, respectively). Moreover, most of the hemoglobin produced by the latter strain was in the insoluble fraction (compare lane 2 of Fig. 1C to lane 5). No signal was generated by either fraction of BL21(DE3) with heme transport genes alone (lanes 1 and 4), indicating that antibody did not cross-react with P. shigelloides heme transport or E. coli proteins.

Analysis of soluble fractions from three experiments indicated that BL21(DE3) with hemoglobin and heme transport genes produced 5.3 times more hemoglobin (standard deviation, ±0.50) than BL21(DE3) with hemoglobin genes alone. These data match the 5.8-fold increase (standard deviation, ±0.58) in average derivative spectrum values from assays of BL21(DE3) with hemoglobin and heme transport genes versus with hemoglobin genes alone.


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Hemoglobin production in BL21(DE3) containing hemoglobin and heme transport genes on the same plasmid.
 
pHB0.0hug contains both hemoglobin and heme transport genes (Fig. 2A; Table 1). Hemoglobin production was assessed in BL21(DE3) transformed with the one-plasmid system (pHB0.0hug, a high-copy-number plasmid) or cotransformed with the two-plasmid system (pHB0.0, a high-copy-number plasmid, and pHUG21, a low-copy-number plasmid). Unlike BL21(DE3) with the two-plasmid system, the strain with the one-plasmid system produced high derivative spectrum values when grown in L broth without EDDA, presumably because of the higher copy number of the heme transport genes on pHB0.0hug. Thus, the strain with the one-plasmid system was grown in L broth and that with the two-plasmid system in L broth with EDDA.


Figure 2
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  		FIG. 2. Hemoglobin production in BL21(DE3) containing the one-plasmid or two-plasmid system. (A) Genetic and partial restriction enzyme map of genes on pHB0.0hug. Genes are indicated with horizontal arrows, and promoters are indicated with vertical bent arrows. Restriction enzyme sites are as follows: B, BamHI; H, HindIII; N, NheI; S, SalI; and W, BsiWI. (B) Immunoblot of soluble (S) and insoluble (I) fractions. Lanes 1 and 3, BL21(DE3) containing the two-plasmid system (pHB0.0, pHUG21); lanes 2 and 4, BL21(DE3) containing the one-plasmid system (pHB0.0hug); lane 5, 0.45 µg of hemoglobin. (C) Densitometry analysis of immunoblots of soluble fractions. Data are from three independent sets of samples analyzed on the same immunoblot; soluble fractions from 0.004 OD unit of cells at a wavelength of 700 nm were loaded. Open circles, hemoglobin control; filled squares, BL21(DE3) containing the two-plasmid system; filled triangles, BL21(DE3) containing the one-plasmid system.


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  		TABLE 1. Bacterial strains and plasmids used in this study

The results of spectrophotometric hemoglobin assays indicated that average derivative spectrum values of both the single- and double-plasmid strains were 0.134, with standard deviations below 0.008. However, when protein fractions were analyzed, BL21(DE3) with the one-plasmid system (Fig. 2B, lane 2) produced more soluble hemoglobin than with the two-plasmid system (lane 1). Spectrophotometric scans were performed on soluble fractions isolated from both strains. Fractions from BL21(DE3) containing the one-plasmid system had an average derivative value 1.36-fold higher (standard deviation, ±0.02) than that from BL21(DE3) with the two-plasmid system, suggesting that BL21(DE3) with the one-plasmid system did indeed produce more soluble holo-hemoglobin.

To estimate the amount of soluble hemoglobin, samples from 0.004 optical density (OD) unit of cells obtained in three independent experiments were analyzed on an immunoblot containing known amounts of hemoglobin. Data points for BL21(DE3) with the two-plasmid system (Fig. 2C) clustered around 0.5 µg, whereas those for BL21(DE3) with the one-plasmid system ranged from 0.6 µg to 0.75 µg. The average amount of soluble hemoglobin produced per 0.004 OD unit was 0.48 µg for BL21(DE3) with the two-plasmid system and 0.65 µg for BL21(DE3) with the one-plasmid system (Table 2). To estimate the amount of hemoglobin produced per ml of culture, the average absorbance of the culture over 0.004 OD unit was multiplied by the average quantity of hemoglobin per 0.004 OD unit. BL21(DE3) with the one-plasmid system produced 244 µg of hemoglobin per ml, whereas BL21(DE3) with the two-plasmid system produced 142 µg per ml (Table 2). BL21(DE3) with the hemoglobin genes alone produced approximately 24 µg of hemoglobin per ml. Thus, BL21(DE3) with the one-plasmid system produced 10 times more soluble hemoglobin than BL21(DE3) with the hemoglobin genes alone. Additional work is needed to determine whether this approach of coexpressing hemoglobin and heme transport genes in E. coli is an economically feasible method of producing recombinant hemoglobin.


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  		TABLE 2. Analysis of hemoglobin production in BL21(DE3) containing various plasmids


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ACKNOWLEDGMENTS
 
This work was supported by grant development funds from the University of Texas of the Permian Basin, National Institutes of Health grant 1 R15 HL079992-01 (D. P. Henderson), NIH grants GM 35649 and HL 47020, and Welch Foundation grant C-612 (J. S. Olson).

We thank Shelley Payne and Elizabeth Wyckoff for their advice concerning the project.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, University of Texas of the Permian Basin, 4901 East University Blvd., Odessa, TX 79762-0001. Phone: (432) 552-2270. Fax: (432) 552-3230. E-mail: henderson_d{at}utpb.edu Back

{triangledown} Published ahead of print on 1 August 2008. Back


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Applied and Environmental Microbiology, September 2008, p. 5854-5856, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01291-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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