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HNOK Proteinlerinin Escherichia Coli’de Üretiminin Optimizasyonu

Yıl 2017, , 86 - 97, 08.12.2017

Nur Başak Sürmeli

https://doi.org/10.17776/csj.363308

Öz

Hemoproteinler canlılarda steroid biyosentezinden solunuma,
sinyalizasyondan ilaç metabolizmasına kadar pek çok farklı biyolojik süreçlerde
önemli görevler üstlenirler. Endüstride, hemoproteinler kolesterol düşürücü
pravastatin, rahim ve rahim ağzı kanserlerinin hormonal tedavisinde kullanılan
progesteron, alerji ve yangı’ya karşı kullanılan kortizon gibi ilaçların
üretiminde kullanılmaktadır. Bunların yanı sıra hemoproteinlerin ilaç
geliştirilmesi, biyolojik iyileştirme gibi alanlarda kullanılması da planlanmaktadır.
Moleküler biyoloji ve protein tasarımı tekniklerinin gelişmesi ile bu
proteinlerin endüstriyel uygulama alanları da genişleyecektir. Hemoproteinlerin
bu alanlarda yaygın kullanımı karşısındaki en önemli engellerden biri Hem
kofaktörüne bağlı bir şekilde yüksek miktarda hemoprotein üretilememesidir. Bu
çalışmada Hem kofaktörüne bağlı bir şekilde üretilen hemoprotein miktarını en
yüksek seviyeye getirebilecek koşullar araştırılmıştır. Bu çalışma kapsamında
bakteride hemoprotein üretimini etkileyen en önemli üç etken olan indükleyici
izopropil β-D-l-tiyogalaktopiranosid (IPTG), ve Hem öncül molekülü
δ-aminolevülinik asit (ALA), ve ekspresyon sıcaklığı incelenmiştir. Bu
etkenlerin termofilik hemoprotein TtHNOK
üretimine etkisi araştırılmıştır. Özellikle, ALA pahalı bir molekül olduğu için
hemoproteinlerin üretiminde kullanılan ALA miktarının optimizasyonu önemlidir.
Bu çalışma sonucunda Escherichia coli
bakterisinde Hem kofaktörüne bağlı TtHNOK proteinin üretimi için en uygun
koşulların düşük sıcaklık, 0,5 mM IPTG ve 1 mM ALA olduğu gösterilmiştir. Bu
çalışmada çıkan sonuçlar Hem kofaktörüne bağlı hemoprotein üretiminde ALA
konsantrasyonun ve ekspresyon sıcaklığının önemli olduğunu göstermiştir. 

Anahtar Kelimeler

Hemoproteinler protein ekspresyonu hem biyosentezi δ-aminolevülinik asit

Kaynakça

  • [1] Lu Y., Berry S.M., Pfister T.D. Engineering novel metalloproteins: Design of metal-binding sites into native protein scaffolds. Chem Rev 2001; 101: 3047-80.
  • [2] Van Beilen J.B., Duetz W.A., Schmid A., Witholt B. Practical issues in the application of oxygenases Trends Biotechnol 2003; 21: 170-7.
  • [3] Hogg J.A., Steroids, the steroid community, and Upjohn in perspective: A profile of innovation. Steroids 1992; 57: 593-616.
  • [4] Peterson D.H., Murray H.C. Microbiological oxygenation of steroids at carbon 11. J Am Chem Soc 1952; 74: 1871-2.
  • [5] Kumar S. Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation. Expert Opin Drug Metab Toxicol 2010; 6: 115-31.
  • [6] Caswell J.M., O’Neill M., Taylor SJC., Moody TS. Engineering and application of P450 monooxygenases in pharmaceutical and metabolite synthesis. Curr Opin Chem Biol 2013; 17: 271-5.
  • [7] Renault H., Bassard J.E., Hamberger B., Werck-Reichhart D. Cytochrome P450-mediated metabolic engineering: current progress and future challenges. Curr Opin Plant Biol 2014; 19C: 27-34.
  • [8] Prabhulkar S., Tian H., Wang X., Zhu J.J., Li C.Z. Engineered Proteins: Redox Properties and Their Applications. Antioxid Redox Signal 2012; 17: 1796-1822.
  • [9] Eggins B.R., Chemical Sensors and Biosensors. West Sussex, England: John Wiley & Sons, Ltd, 2002.
  • [10] Koder R.L., Anderson J.L.R., Solomon L.A., Reddy K.S., Moser C.C., Dutton P.L. Design and engineering of an O2 transport protein. Nature 2009; 458: 305-9
  • [11] Springer B.A., Sligar S.G. High-level expression of sperm whale myoglobin in Escherichia coli. Proc Natl Acad Sci USA 1987; 84: 8961-5.
  • [12] Chudaev M.V., Usanov S.A. Expression of functionally active cytochrome b5 in Escherichia coli: isolation, purification, and use of the immobilized recombinant heme protein for affinity chromatography of electron-transfer proteins. Biochemistry (Mosc) 1997; 62: 401-11.
  • [13] Choby J.E., Skaar E.P. Heme Synthesis and Acquisition in Bacterial Pathogens. J Mol Biol 2016; 428: 3408-3428.
  • [14] Harnastai I.N., Gilep A.A., Usanov S.A. The development of an efficient system for heterologous expression of cytochrome P450s in Escherichia coli using hemA gene co-expression. Protein Expr Purif 2006; 46: 47-55.
  • [15] Harris W.F., Burkhalter R.S., Lin W., Timkovich R. Enhancement of Bacterial Porphyrin Biosynthesis by Exogenous Aminolevulinic Acid and Isomer Specificity of the Products. Bioorg Chem 1993; 21: 209-20.
  • [16] Iyer L.M., Anantharaman V., Aravind L. Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins. BMC Genomics 2003; 4: 5.
  • [17] Karow D.S., Pan D., Tran R., Pellicena P., Presley A., Mathies R.A., Marletta M.A. Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis. Biochemistry 2004; 43: 10203-11.
  • [18] Plate L., Marletta M.A. Nitric oxide-sensing H-NOX proteins govern bacterial communal behavior. Trends Biochem Sci 2013; 38: 566-575.
  • [19] Boon E.M., Marletta M.A. Ligand specificity of H-NOX domains: From sGC to bacterial NO sensors J Inorg Biochem 2005; 99: 892-902.
  • [20] Xue Y., Xu Y., Liu Y., Ma Y., Zhou P. Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int J Syst Evol Microbiol 2001; 51: 1335-41.
  • [21] Olea C., Kuriyan J., Marletta M.A. Modulating heme redox potential through protein-induced porphyrin distortion. J Am Chem Soc 2010; 132: 12794-5.
  • [22] Weinert E.E., Phillips-Piro C.M., Marletta M.A. Porphyrin π-stacking in a heme protein scaffold tunes gas ligand affinity. J Inorg Biochem 2013; 127: 7-12.
  • [23] Azarov I., Wang L., Rose JJ., Xu Q., Huang X.N., Belanger A., Wang Y., Guo L., Liu C., Ucer K.B., McTiernan C.F, ODonnell C.P., Shiva S., Tejero J., Kim-Shapiro D.B., Gladwin M.T. Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning. Sci Transl Med 2016; 8: 368ra173-368ra173.
  • [24] Winter M.B., Klemm P.J., Phillips-Piro C.M., Raymond K.N., Marletta M.A. Porphyrin-Substituted H-NOX Proteins as High-Relaxivity MRI Contrast Agents. Inorg Chem 2013; 52: 2277-2279.
  • [25] Nierth A., Marletta M.A. Direct meso-alkynylation of metalloporphyrins through gold catalysis for hemoprotein engineering. Angew Chemie Int Ed 2014; 53: 2611-4.
  • [26] Weinert E.E., Plate L., Whited C.A., Olea C., Marletta M.A. Determinants of ligand affinity and heme reactivity in H-NOX domains. Angew Chemie Int Ed 2010; 49: 720-723.
  • [27] Zhao Y., Marletta M.A. Localization of the heme binding region in soluble guanylate cyclase. Biochemistry 1997; 36: 15959-64.
  • [28] Green M.R., Sambrook J. Molecular Cloning A Laboratory Manual. 4th ed Cold Spring Harbor laboratory Press, 2014.
  • [29] Ayato Y., Matsuda N. Evaluation of biofuel cells with hemoglobin as cathodic electrocatalysts for hydrogen peroxide reduction on bare indium-tin-oxide electrodes. Energies 2014; 7: 1-12.
  • [30] Stone K., Ahmed S. Advances in Engineered Hemoproteins that Promote Biocatalysis. Inorganics 2016; 4: 12.
  • [31] Hernandez K.E., Renata H., Lewis RD., Kan S.B.J., Zhang C., Forte J., Rozzell D., McIntosh J.A., Arnold F.H. Highly Stereoselective Biocatalytic Synthesis of Key Cyclopropane Intermediate to Ticagrelor. ACS Catal 2016; 6: 7810-3
  • [32] Kafi A.K.M., Lee D.Y., Park S.H., Kwon Y.S. Electrochemical properties of heme-protein in lauric acid films and its application as a biosensor. NanoBiotechnology 2006; 2: 67-70.
  • [33] Hofbauer S., Schaffner I., Furtmüller P.G., Obinger C. Chlorite dismutases - a heme enzyme family for use in bioremediation and generation of molecular oxygen. Biotechnol J 2014; 9: 461-473.
  • [34] Shaik T.B., Pal B. Fine Tuning Soluble Expression of a Heme Protein. J. Proteins Proteomics 2015; 6: 255-60.
  • [35] Jung Y., Kwak J., Lee Y. High-level production of heme-containing holoproteins in Escherichia coli. Appl Microbiol Biotechnol 2001; 55: 187-91.
  • [36] Hayashi T., Takimura T., Aoyama Y., Hitomi Y., Suzuki A., Ogoshi H. Structure and reactivity of reconstituted myoglobins: interaction between protein and polar side chain of chemically modified hemin. Inorganica Chim Acta 1998; 275-276: 159-67.
  • [37] Hargrove M.S., Krzywda S., Wilkinson A.J., Dou Y., Ikeda-Saito M., Olson J.S. Stability of myoglobin: a model for the folding of heme proteins. Biochemistry 1994; 33: 11767-75.
  • [38] Correia M.A. Its Heme and Apoprotein Moieties in Synthesis, assembly, repair and disposal. Drug Metab Rev 2011; 43: 1-26.

Optimization of Hnox Protein Production in Escherichia Coli

Yıl 2017, , 86 - 97, 08.12.2017

Nur Başak Sürmeli

https://doi.org/10.17776/csj.363308

Öz

Hemeproteins
carry a variety of different functions in organisms ranging from steroid
biosynthesis to respiration, signaling to drug metabolism. In industry,
hemeproteins are used for production of drugs such as: pravastatin for lowering
cholesterol, progesterone for hormonal treatment of cancers of uterus and
cervix, and cortisone used against allergy and inflammation. Hemeproteins can
also be used in drug development and biological remediation. The industrial
applications of hemeproteins will expand with development of molecular biology
and protein design techniques. One of the obstacles to the widespread use of
hemeproteins is difficulties in production of high levels of heme bound
protein. This study aims to maximize the amount of heme cofactor bound hemeprotein
produced. Here, three important factors affecting hemeprotein production in
bacteria are examined: induction by isopropyl β-D-1-thiogalactopyranoside
(IPTG), δ-aminolevulinic acid (ALA), precursor for heme biosynthesis, and
expression temperature. Effects of these factors on production of thermophilic
hemeprotein TtHNOX are investigated.
Since ALA is an expensive molecule, optimization of the amount of ALA used is
important. The most suitable conditions to produce TtHNOX is at low temperature, 0.5 mM IPTG and 1 mM ALA. This study
concludes that ALA concentration and expression temperature are important in
production of heme bound hemeproteins.

Anahtar Kelimeler

Hemeproteins protein expression heme biosynthesis δ-aminolevulinic acid

Kaynakça

  • [1] Lu Y., Berry S.M., Pfister T.D. Engineering novel metalloproteins: Design of metal-binding sites into native protein scaffolds. Chem Rev 2001; 101: 3047-80.
  • [2] Van Beilen J.B., Duetz W.A., Schmid A., Witholt B. Practical issues in the application of oxygenases Trends Biotechnol 2003; 21: 170-7.
  • [3] Hogg J.A., Steroids, the steroid community, and Upjohn in perspective: A profile of innovation. Steroids 1992; 57: 593-616.
  • [4] Peterson D.H., Murray H.C. Microbiological oxygenation of steroids at carbon 11. J Am Chem Soc 1952; 74: 1871-2.
  • [5] Kumar S. Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation. Expert Opin Drug Metab Toxicol 2010; 6: 115-31.
  • [6] Caswell J.M., O’Neill M., Taylor SJC., Moody TS. Engineering and application of P450 monooxygenases in pharmaceutical and metabolite synthesis. Curr Opin Chem Biol 2013; 17: 271-5.
  • [7] Renault H., Bassard J.E., Hamberger B., Werck-Reichhart D. Cytochrome P450-mediated metabolic engineering: current progress and future challenges. Curr Opin Plant Biol 2014; 19C: 27-34.
  • [8] Prabhulkar S., Tian H., Wang X., Zhu J.J., Li C.Z. Engineered Proteins: Redox Properties and Their Applications. Antioxid Redox Signal 2012; 17: 1796-1822.
  • [9] Eggins B.R., Chemical Sensors and Biosensors. West Sussex, England: John Wiley & Sons, Ltd, 2002.
  • [10] Koder R.L., Anderson J.L.R., Solomon L.A., Reddy K.S., Moser C.C., Dutton P.L. Design and engineering of an O2 transport protein. Nature 2009; 458: 305-9
  • [11] Springer B.A., Sligar S.G. High-level expression of sperm whale myoglobin in Escherichia coli. Proc Natl Acad Sci USA 1987; 84: 8961-5.
  • [12] Chudaev M.V., Usanov S.A. Expression of functionally active cytochrome b5 in Escherichia coli: isolation, purification, and use of the immobilized recombinant heme protein for affinity chromatography of electron-transfer proteins. Biochemistry (Mosc) 1997; 62: 401-11.
  • [13] Choby J.E., Skaar E.P. Heme Synthesis and Acquisition in Bacterial Pathogens. J Mol Biol 2016; 428: 3408-3428.
  • [14] Harnastai I.N., Gilep A.A., Usanov S.A. The development of an efficient system for heterologous expression of cytochrome P450s in Escherichia coli using hemA gene co-expression. Protein Expr Purif 2006; 46: 47-55.
  • [15] Harris W.F., Burkhalter R.S., Lin W., Timkovich R. Enhancement of Bacterial Porphyrin Biosynthesis by Exogenous Aminolevulinic Acid and Isomer Specificity of the Products. Bioorg Chem 1993; 21: 209-20.
  • [16] Iyer L.M., Anantharaman V., Aravind L. Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins. BMC Genomics 2003; 4: 5.
  • [17] Karow D.S., Pan D., Tran R., Pellicena P., Presley A., Mathies R.A., Marletta M.A. Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis. Biochemistry 2004; 43: 10203-11.
  • [18] Plate L., Marletta M.A. Nitric oxide-sensing H-NOX proteins govern bacterial communal behavior. Trends Biochem Sci 2013; 38: 566-575.
  • [19] Boon E.M., Marletta M.A. Ligand specificity of H-NOX domains: From sGC to bacterial NO sensors J Inorg Biochem 2005; 99: 892-902.
  • [20] Xue Y., Xu Y., Liu Y., Ma Y., Zhou P. Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int J Syst Evol Microbiol 2001; 51: 1335-41.
  • [21] Olea C., Kuriyan J., Marletta M.A. Modulating heme redox potential through protein-induced porphyrin distortion. J Am Chem Soc 2010; 132: 12794-5.
  • [22] Weinert E.E., Phillips-Piro C.M., Marletta M.A. Porphyrin π-stacking in a heme protein scaffold tunes gas ligand affinity. J Inorg Biochem 2013; 127: 7-12.
  • [23] Azarov I., Wang L., Rose JJ., Xu Q., Huang X.N., Belanger A., Wang Y., Guo L., Liu C., Ucer K.B., McTiernan C.F, ODonnell C.P., Shiva S., Tejero J., Kim-Shapiro D.B., Gladwin M.T. Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning. Sci Transl Med 2016; 8: 368ra173-368ra173.
  • [24] Winter M.B., Klemm P.J., Phillips-Piro C.M., Raymond K.N., Marletta M.A. Porphyrin-Substituted H-NOX Proteins as High-Relaxivity MRI Contrast Agents. Inorg Chem 2013; 52: 2277-2279.
  • [25] Nierth A., Marletta M.A. Direct meso-alkynylation of metalloporphyrins through gold catalysis for hemoprotein engineering. Angew Chemie Int Ed 2014; 53: 2611-4.
  • [26] Weinert E.E., Plate L., Whited C.A., Olea C., Marletta M.A. Determinants of ligand affinity and heme reactivity in H-NOX domains. Angew Chemie Int Ed 2010; 49: 720-723.
  • [27] Zhao Y., Marletta M.A. Localization of the heme binding region in soluble guanylate cyclase. Biochemistry 1997; 36: 15959-64.
  • [28] Green M.R., Sambrook J. Molecular Cloning A Laboratory Manual. 4th ed Cold Spring Harbor laboratory Press, 2014.
  • [29] Ayato Y., Matsuda N. Evaluation of biofuel cells with hemoglobin as cathodic electrocatalysts for hydrogen peroxide reduction on bare indium-tin-oxide electrodes. Energies 2014; 7: 1-12.
  • [30] Stone K., Ahmed S. Advances in Engineered Hemoproteins that Promote Biocatalysis. Inorganics 2016; 4: 12.
  • [31] Hernandez K.E., Renata H., Lewis RD., Kan S.B.J., Zhang C., Forte J., Rozzell D., McIntosh J.A., Arnold F.H. Highly Stereoselective Biocatalytic Synthesis of Key Cyclopropane Intermediate to Ticagrelor. ACS Catal 2016; 6: 7810-3
  • [32] Kafi A.K.M., Lee D.Y., Park S.H., Kwon Y.S. Electrochemical properties of heme-protein in lauric acid films and its application as a biosensor. NanoBiotechnology 2006; 2: 67-70.
  • [33] Hofbauer S., Schaffner I., Furtmüller P.G., Obinger C. Chlorite dismutases - a heme enzyme family for use in bioremediation and generation of molecular oxygen. Biotechnol J 2014; 9: 461-473.
  • [34] Shaik T.B., Pal B. Fine Tuning Soluble Expression of a Heme Protein. J. Proteins Proteomics 2015; 6: 255-60.
  • [35] Jung Y., Kwak J., Lee Y. High-level production of heme-containing holoproteins in Escherichia coli. Appl Microbiol Biotechnol 2001; 55: 187-91.
  • [36] Hayashi T., Takimura T., Aoyama Y., Hitomi Y., Suzuki A., Ogoshi H. Structure and reactivity of reconstituted myoglobins: interaction between protein and polar side chain of chemically modified hemin. Inorganica Chim Acta 1998; 275-276: 159-67.
  • [37] Hargrove M.S., Krzywda S., Wilkinson A.J., Dou Y., Ikeda-Saito M., Olson J.S. Stability of myoglobin: a model for the folding of heme proteins. Biochemistry 1994; 33: 11767-75.
  • [38] Correia M.A. Its Heme and Apoprotein Moieties in Synthesis, assembly, repair and disposal. Drug Metab Rev 2011; 43: 1-26.
Toplam 38 adet kaynakça vardır.

Ayrıntılar

Bölüm Natural Sciences
Yazarlar

Nur Başak Sürmeli

Yayımlanma Tarihi 8 Aralık 2017
Gönderilme Tarihi 1 Temmuz 2017
Kabul Tarihi 21 Kasım 2017
Yayımlandığı Sayı Yıl 2017

Kaynak Göster

APA Sürmeli, N. B. (2017). Optimization of Hnox Protein Production in Escherichia Coli. Cumhuriyet Science Journal, 38(4), 86-97. https://doi.org/10.17776/csj.363308