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Recombinant Hemoglobin II From Lucina pectinata: A Large-Scale Method For Hemeprotein Expression in E. coli

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Abstract

Hemoglobin II from the clam L. pectinata is an O2 reactive protein that remains oxygenated in the presence of other molecules. To determine the mechanism of ligand selection in this hemoglobin, rHbII was expressed in large quantities using an improved fermentation process. The highest protein yield was obtained by: transforming HbII into the BLi5 cells, inducing and supplementing the culture during the mid-log phase with 1 mM IPTG, 30 μg/mL hemin chloride and 1% glucose, and decreasing the temperature to 30 °C after induction. In addition, cell culture density was greatly enhanced by using glycerol, adding MgSO4, supplementing the media with glucose after the glycerol was consumed and maintaining the dissolved oxygen at 35%. Under these conditions the maximum protein yield obtained was ~2,300 mg/L. The results indicate that rHbII is similar to the native protein. The protocol was validated with other hemoglobins, indicating that it can be extended to other hemeproteins.

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Abbreviations

HbI, HbII, and HbIII:

Hemoglobins I, II and III

rHbII:

Recombinant HbII

nHbII:

Native HbII

L. pectinata :

Lucina pectinata

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

k on :

Association rate constant

k off :

Dissociation rate constant

Gln:

Glutamine

Phe:

Phenylalanine

Tyr:

Tyrosine

RT-PCR:

Reverse transcription polymerase chain reaction

TB:

Terrific broth

E. coli :

Escherichia coli

cDNA:

Complementary DNA

OD:

Optical density

dO2 :

Dissolved oxygen

References

  1. Aristidou AA, San KY, Bennett GN (1995) Biotechnol Prog 11:475–478

    Article  CAS  Google Scholar 

  2. Bradford MM (1979) Anal Biochem 72:248–254

    Article  Google Scholar 

  3. Chow DC, Dreher MR, Trabbic-Carlson K, Chilkoti A (2006) Biotechnol Prog 22:638–646

    Article  CAS  Google Scholar 

  4. Dantsker D, Roche C, Samuni U, Blouin G, Olson JS, Friedman JM (2005) J Biol Chem 280:38740–38755

    Article  CAS  Google Scholar 

  5. De Jesús-Bonilla W, Jia Y, Alayash AI, López-Garriga J (2007) Biochemistry 46:10451–10460

    Article  Google Scholar 

  6. Draghi F, Miele AE, Travaglini-Allocatelli C, Vallone B, Brunori M, Gibson QH, Olson JS (2002) J Biol Chem 277:7509–7519

    Article  CAS  Google Scholar 

  7. Egawa T, Yeh S-R (2005) J Inor Biochem 99:72–96

    Article  CAS  Google Scholar 

  8. Eiteman MA, Altman E (2006) Trends Biotechnol 24:530–536

    Article  CAS  Google Scholar 

  9. Gavira JA, Camara-Artigas A, De Jesús-Bonilla W, López-Garriga J, Lewis A, Pietri R, Yeh S-R, Cadilla CL, García-Ruiz JM (2008) J Biol Chem 283:9414–9423

    Article  CAS  Google Scholar 

  10. Graves PE, Henderson DP, Horstman MJ, Solomon BJ, Olson JS (2008) Biochim Biophys Acta 1784:471–479

    Google Scholar 

  11. Hoo H, Hashidoko Y, Islam MT, Tahara S (2004) Appl Environ Microbiol 70:5214–5221

    Article  CAS  Google Scholar 

  12. Kraus DW, Wittenberg JB (1990) J Biol Chem 265:16043–16053

    CAS  Google Scholar 

  13. Kraus DW, Wittenberg JB (1990) J Biol Chem 265:16054–16059

    CAS  Google Scholar 

  14. León RG, Munier-Lehmann H, Barzu O, Baudin-Creuza V, Pietri R, López-Garriga J, Cadilla CL (2004) Protein Expr Purif 38:184–195

    Google Scholar 

  15. Liu T, Chen JY, Zheng Z, Wang TH, Chen GQ (2005) Appl Microbiol Biotechnol 68:346–354

    Article  CAS  Google Scholar 

  16. Maillett DH, Simplaceanu V, Shen TJ, Ho NT, Olson JS, Ho C (2008) Biochemistry 47:10551–10563

    Article  CAS  Google Scholar 

  17. Olson JS, Soman J, Phillips GN Jr (2007) IUBMB Life 59:552–562

    Article  CAS  Google Scholar 

  18. Pietri R, León RG, Kiger L, Marden MC, Granell LB, Cadilla CL, López-Garriga J (2006) Biochim Biophys Acta 1764:758–765

    CAS  Google Scholar 

  19. Pietri R, Lewis A, León RG, Casabona G, Kiger L, Yeh SR, Fernandez-Alberti S, Marden MC, Cadilla CL, López-Garriga J (2009) Biochemistry 48:4881–4894

    Article  CAS  Google Scholar 

  20. Pflug S, Richter SM, Urlacher VB (2007) J Biotechnol 129:481–488

    Article  CAS  Google Scholar 

  21. Rivera L, López-Garriga J, Cadilla CL (2008) Gene 410:122–128

    Article  CAS  Google Scholar 

  22. Rizzi M, Wittenberg JB, Coda A, Ascenzi P, Bolognesi M (1996) J Mol Biol 258:1–5

    Article  CAS  Google Scholar 

  23. Springer BA, Egeberg KD, Sligar SG, Rohlfs RJ, Mathews AJ, Olson JS (1989) J Biol Chem 264:3057–3060

    CAS  Google Scholar 

  24. Studier FW (2005) Protein Expr Purif 41:207–234

    Article  CAS  Google Scholar 

  25. Torres-Mercado E, Renta JY, Rodríguez Y, López-Garriga J, Cadilla CL (2003) J Protein Chem 22:683–690

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by funds from the National Science Foundation, Cellular Biology (Grant 0544250), NIH-NIGMS/MBRS-SCORE S06GM08103-34 and NCRR RCMI grant G12RR03051. We thank Dr. Ruth G. Leon for the construction of the HbII pET-28a(+) vector. C. R. also thanks the Alfred P. Sloan (NACME) and CNY-PR AGEP for their fellowship support.

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Authors and Affiliations

  1. Department of Chemistry, University of Puerto Rico, Mayagüez Campus, PO BOX 9019, Mayagüez, PR, 00681-9019, USA

    Cacimar Ramos, Ruth Pietri, Wilmarie Lorenzo, Elddie Roman, Laura B. Granell & Juan López-Garriga

  2. Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, PO BOX 365067, San Juan, PR, 00936-5067, USA

    Carmen L. Cadilla

Authors
  1. Cacimar Ramos
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  2. Ruth Pietri
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  3. Wilmarie Lorenzo
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  4. Elddie Roman
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  5. Laura B. Granell
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  6. Carmen L. Cadilla
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  7. Juan López-Garriga
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Corresponding author

Correspondence to Juan López-Garriga.

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Cacimar Ramos and Wilmarie Lorenzo contributed equally to this work.

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Ramos, C., Pietri, R., Lorenzo, W. et al. Recombinant Hemoglobin II From Lucina pectinata: A Large-Scale Method For Hemeprotein Expression in E. coli . Protein J 29, 143–151 (2010). https://doi.org/10.1007/s10930-010-9234-8

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  • DOI: https://doi.org/10.1007/s10930-010-9234-8

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