Login Register Cart 0
Generic placeholder image

Protein & Peptide Letters

Editor-in-Chief

ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Process Development for the Production and Purification of PEGylated RhG-CSF Expressed in Escherichia coli

Author(s): Zeinab Mohammadi*, Mahdi Alijanianzadeh, Rassoul Khalilzadeh and Sirus Khodadadi

Volume 29, Issue 4, 2022

Published on: 31 March, 2022

Page: [293 - 305] Pages: 13

DOI: 10.2174/0929866529666220126100559

Price: $65

Abstract

Background and Objectives: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) and its PEGylated form (PEG-GCSF) are used in cancer therapy. Thus, developing a more cost-effectively method for expressing rhG-CSF and the PEGylation optimization of rhG-CSF by reaction engineering and subsequent purification strategy is necessary.

Methods: RhG-CSF expression in Escherichia coli BL21 (DE3) was carried out by auto-induction batch fermentation and improved for maximizing rhG-CSF productivity. After that, purified rhGCSF was PEGylated using methoxy polyethylene glycol propionaldehydes (mPEG20-ALD). The various conditions effect of extraction and purification of rhG-CSF and PEG-GCSF were assayed.

Results: The assessment results revealed that the auto-induction batch cultivation strategy had maximum productivity, and rhG-CSF purity was more than 99%. The obtained data of rhG-CSF PEGylation displayed that the optimized conditions of rhG-CSF PEGylation and purification enhanced homogeneity PEG-GCSF and managed reaction toward optimal yield of PEG-GCSF (70%) and purity of 99.9%. Findings from FTIR, CD, fluorescence spectroscopy, and bioassay revealed that PEGylation was executed exactly in the rhG-CSF N-terminus, and products maintained their conformation properties.

Conclusion: Overall, the developed approach expanded strategies for high yield rhG-CSF by simplified auto-induction batch fermentation system and rhG-CSF PEGylation, which are simple and timesaving, economical, and high efficiency.

Keywords: Auto-induction, batch fermentation, Escherichia coli, PEGylation, process development, RhG-CSF.

« Previous Next »
Graphical Abstract

[1]
Qi, Y.; Chilkoti, A. Protein-polymer conjugation-moving beyond PEGylation. Curr. Opin. Chem. Biol., 2015, 28, 181-193.
[http://dx.doi.org/10.1016/j.cbpa.2015.08.009] [PMID: 26356631]
[2]
Turecek, P.L.; Bossard, M.J.; Schoetens, F.; Ivens, I.A. PEGylation of biopharmaceuticals: A review of chemistry and nonclinical safety information of approved drugs. J. Pharm. Sci., 2016, 105(2), 460-475.
[http://dx.doi.org/10.1016/j.xphs.2015.11.015] [PMID: 26869412]
[3]
Tripathi, N.K.; Shrivastava, A. Recent developments in bioprocessing of recombinant proteins: expression hosts and process development. Front. Bioeng. Biotechnol., 2019, 7, 420.
[http://dx.doi.org/10.3389/fbioe.2019.00420] [PMID: 31921823]
[4]
Gascon, P. Presently available biosimilars in hematology-oncology: G-CSF. Target. Oncol., 2012, 7(1)(Suppl. 1), S29-S34.
[http://dx.doi.org/10.1007/s11523-011-0190-9] [PMID: 22258705]
[5]
Oliveira, C.; Domingues, L. Guidelines to reach high-quality purified recombinant proteins. Appl. Microbiol. Biotechnol., 2018, 102(1), 81-92.
[http://dx.doi.org/10.1007/s00253-017-8623-8] [PMID: 29151158]
[6]
Spencer, B.W.; Williams, H.R.; Gee, S.H.; Whiskey, E.; Rodrigues, J.P.; Mijovic, A.; MacCabe, J.H. Granulocyte colony stimulating factor (G-CSF) can allow treatment with clozapine in a patient with severe benign ethnic neutropaenia (BEN): a case report. J. Psychopharmacol., 2012, 26(9), 1280-1282.
[http://dx.doi.org/10.1177/0269881112450782] [PMID: 22719015]
[7]
Bendall, L.J.; Bradstock, K.F.G-C.SF. From granulopoietic stimulant to bone marrow stem cell mobilizing agent. Cytokine Growth Factor Rev., 2014, 25(4), 355-367.
[http://dx.doi.org/10.1016/j.cytogfr.2014.07.011] [PMID: 25131807]
[8]
Panopoulos, A.D.; Watowich, S.S. Granulocyte colony-stimulating factor: molecular mechanisms of action during steady state and ‘emergency’ hematopoiesis. Cytokine, 2008, 42(3), 277-288.
[http://dx.doi.org/10.1016/j.cyto.2008.03.002] [PMID: 18400509]
[9]
Bashir, S; Sadaf, S.; Ahmad, S; Akhtar, MW Enhanced and secretory expression of human granulocyte colony stimulating factor by Bacillus subtilis SCK6. BioMed Res. Int., 2015, 2015, 636249.
[10]
Kim, C.K.; Lee, C.H.; Lee, S-B.; Oh, J-W. Simplified large-scale refolding, purification, and characterization of recombinant human granulocyte-colony stimulating factor in Escherichia coli. PLoS One, 2013, 8(11), e80109.
[http://dx.doi.org/10.1371/journal.pone.0080109] [PMID: 24224041]
[11]
Vanz, A.L.; Renard, G.; Palma, M.S.; Chies, J.M.; Dalmora, S.L.; Basso, L.A.; Santos, D.S. Human granulocyte colony stimulating factor (hG-CSF): Cloning, overexpression, purification and characterization. Microb. Cell Fact., 2008, 7(1), 13.
[http://dx.doi.org/10.1186/1475-2859-7-13] [PMID: 18394164]
[12]
Hill, C.P.; Osslund, T.D.; Eisenberg, D. The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. Proc. Natl. Acad. Sci. USA, 1993, 90(11), 5167-5171.
[http://dx.doi.org/10.1073/pnas.90.11.5167] [PMID: 7685117]
[13]
Toghraie, F.S.; Yazdanpanah-Samani, M.; Mahmoudi Maymand, E.; Hosseini, A.; Asgari, A.; Ramezani, A.; Ghaderi, A. Molecular cloning, expression and purification of G-CSF isoform D, an alternative splice variant of human G-CSF. Iran. J. Allergy Asthma Immunol., 2019, 18(4), 419-426.
[PMID: 31522450]
[14]
Mishra, P.; Nayak, B.; Dey, R. PEGylation in anti-cancer therapy: An overview. Asian J. Pharm. Sci., 2016, 11(3), 337-348.
[http://dx.doi.org/10.1016/j.ajps.2015.08.011]
[15]
Pfister, D.; Morbidelli, M. Process for protein PEGylation. J. Control. Release, 2014, 180, 134-149.
[http://dx.doi.org/10.1016/j.jconrel.2014.02.002] [PMID: 24531008]
[16]
Suk, J.S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L.M. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery., Adv. Drug Deliv. Rev., 2016, 99(Pt A), 28-51..
[http://dx.doi.org/10.1016/j.addr.2015.09.012] [PMID: 26456916]
[17]
Gefen, T.; Vaya, J.; Khatib, S.; Harkevich, N.; Artoul, F.; Heller, E.D.; Pitcovski, J.; Aizenshtein, E. The impact of PEGylation on protein immunogenicity. Int. Immunopharmacol., 2013, 15(2), 254-259.
[http://dx.doi.org/10.1016/j.intimp.2012.12.012] [PMID: 23306102]
[18]
Alconcel, S.N.; Baas, A.S.; Maynard, H.D. FDA-approved poly (ethylene glycol)-protein conjugate drugs. Polym. Chem., 2011, 2(7), 1442-1448.
[http://dx.doi.org/10.1039/c1py00034a]
[19]
Molineux, G. The design and development of pegfilgrastim (PEG-rmetHuG-CSF, Neulasta). Curr. Pharm. Des., 2004, 10(11), 1235-1244.
[http://dx.doi.org/10.2174/1381612043452613] [PMID: 15078138]
[20]
Pasut, G.; Veronese, F.M. State of the art in PEGylation: The great versatility achieved after forty years of research. J. Control. Release, 2012, 161(2), 461-472.
[http://dx.doi.org/10.1016/j.jconrel.2011.10.037] [PMID: 22094104]
[21]
Morishita, M.; Leonard, R.C. Pegfilgrastim; a neutrophil mediated granulocyte colony stimulating factor-expanding uses in cancer chemotherapy. Expert Opin. Biol. Ther., 2008, 8(7), 993-1001.
[http://dx.doi.org/10.1517/14712598.8.7.993] [PMID: 18549328]
[22]
Pelegri-O’Day, E.M.; Lin, E-W.; Maynard, H.D. Therapeutic protein-polymer conjugates: Advancing beyond PEGylation. J. Am. Chem. Soc., 2014, 136(41), 14323-14332.
[http://dx.doi.org/10.1021/ja504390x] [PMID: 25216406]
[23]
Malekian, R.; Jahanian-Najafabadi, A.; Moazen, F.; Ghavimi, R.; Mohammadi, E.; Akbari, V. High-yield production of granulocyte-macrophage colony-stimulating factor in E. coli BL21 (DE3) By an auto-induction strategy. Iran. J. Pharm. Res., 2019, 18(1), 469-478.
[PMID: 31089381]
[24]
Kittler, S.; Kopp, J.; Veelenturf, P.G.; Spadiut, O.; Delvigne, F.; Herwig, C.; Slouka, C. The lazarus Escherichia coli effect: Recovery of productivity on glycerol/lactose mixed feed in continuous biomanufacturing. Front. Bioeng. Biotechnol., 2020, 8, 993.
[http://dx.doi.org/10.3389/fbioe.2020.00993] [PMID: 32903513]
[25]
Grabski, A.; Mehler, M.; Drott, D. The overnight express autoinduction system: high-density cell growth and protein expression while you sleep. Nat. Methods, 2005, 2(3), 233-235.
[http://dx.doi.org/10.1038/nmeth0305-233]
[26]
Fathi-Roudsari, M.; Maghsoudi, N.; Maghsoudi, A.; Niazi, S.; Soleiman, M. Auto-induction for high level production of biologically active reteplase in Escherichia coli. Protein Expr. Purif., 2018, 151, 18-22.
[http://dx.doi.org/10.1016/j.pep.2018.05.008] [PMID: 29775664]
[27]
Hausjell, J.; Weissensteiner, J.; Molitor, C.; Halbwirth, H.; Spadiut, O.E. coli HMS174(DE3) is a sustainable alternative to BL21(DE3). Microb. Cell Fact., 2018, 17(1), 169.
[http://dx.doi.org/10.1186/s12934-018-1016-6] [PMID: 30376846]
[28]
Bishop, B.; Koay, D.C.; Sartorelli, A.C.; Regan, L. Reengineering granulocyte colony-stimulating factor for enhanced stability. J. Biol. Chem., 2001, 276(36), 33465-33470.
[http://dx.doi.org/10.1074/jbc.M104494200] [PMID: 11406632]
[29]
Kuga, T.; Komatsu, Y.; Yamasaki, M.; Sekine, S.; Miyaji, H.; Nishi, T.; Sato, M.; Yokoo, Y.; Asano, M.; Okabe, M. Mutagenesis of human granulocyte colony stimulating factor. Biochem. Biophys. Res. Commun., 1989, 159(1), 103-111.
[http://dx.doi.org/10.1016/0006-291X(89)92410-8] [PMID: 2466458]
[30]
Wingfield, P.; Benedict, R.; Turcatti, G.; Allet, B.; Mermod, J.J.; DeLamarter, J.; Simona, M.G.; Rose, K. Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF). Biochem. J., 1988, 256(1), 213-218.
[http://dx.doi.org/10.1042/bj2560213] [PMID: 2464993]
[31]
Fallah, M.; Akbari, B.; Saeedinia, A.; Karimi, M.; Vaez, M.; Zeinoddini, M.; Soleimani, M.; Maghsoudi, N. Overexpression of recombinant human granulocyte colony-stimulating factor in E. coli. Iran. J. Med. Sci., 2015, 28(3), 131-134.
[32]
Choi, J.H.; Keum, K.C.; Lee, S.Y. Production of recombinant proteins by high cell density culture of Escherichia coli. Chem. Eng. Sci., 2006, 61(3), 876-885.
[http://dx.doi.org/10.1016/j.ces.2005.03.031]
[33]
Mulkerrin, M.G.; Wetzel, R. pH dependence of the reversible and irreversible thermal denaturation of gamma interferons. Biochemistry, 1989, 28(16), 6556-6561.
[http://dx.doi.org/10.1021/bi00442a005] [PMID: 2506928]
[34]
Natalello, A.; Ami, D.; Collini, M.; D’Alfonso, L.; Chirico, G.; Tonon, G.; Scaramuzza, S.; Schrepfer, R.; Doglia, S.M. Biophysical characterization of Met-G-CSF: effects of different site-specific mono-pegylations on protein stability and aggregation. PLoS One, 2012, 7(8), e42511.
[http://dx.doi.org/10.1371/journal.pone.0042511] [PMID: 22905140]
[35]
Peng, F.; Liu, Y.; Li, X.; Sun, L.; Zhao, D.; Wang, Q.; Ma, G.; Su, Z. PEGylation of G-CSF in organic solvent markedly increase the efficacy and reactivity through protein unfolding, hydrolysis inhibition and solvent effect. J. Biotechnol., 2014, 170, 42-49.
[http://dx.doi.org/10.1016/j.jbiotec.2013.10.037] [PMID: 24315970]
[36]
Son, J.P.; Jun, S-W.; Choi, Y-K.; Park, H.S.; Son, M.K.; Lee, M.Y.; Kang, S.H.; Kang, J.S.; Park, Y.I. Structural identification and biological activity of positional isomers of long-acting and mono-PEGylated recombinant human granulocyte colony-stimulating factor with trimeric-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group. Anal. Biochem., 2012, 423(2), 286-293.
[http://dx.doi.org/10.1016/j.ab.2011.12.014] [PMID: 22244804]
[37]
Zhai, Y.; Zhao, Y.; Lei, J.; Su, Z.; Ma, G. Enhanced circulation half-life of site-specific PEGylated rhG-CSF: Optimization of PEG molecular weight. J. Biotechnol., 2009, 142(3-4), 259-266.
[http://dx.doi.org/10.1016/j.jbiotec.2009.05.012] [PMID: 19497340]
[38]
Seeger, A.; Schneppe, B.; McCarthy, J.E.; Deckwer, W-D.; Rinas, U. Comparison of temperature-and isopropyl-β-D-thiogalacto-pyranoside-induced synthesis of basic fibroblast growth factor in high- cell-density cultures of recombinant Escherichia coli. Enzyme Microb. Technol., 1995, 17(10), 947-953.
[http://dx.doi.org/10.1016/0141-0229(94)00123-9]
[39]
Riesenberg, D.; Guthke, R. High-cell-density cultivation of microorganisms. Appl. Microbiol. Biotechnol., 1999, 51(4), 422-430.
[http://dx.doi.org/10.1007/s002530051412] [PMID: 10341426]
[40]
Lee, S.Y. High cell-density culture of Escherichia coli. Trends Biotechnol., 1996, 14(3), 98-105.
[http://dx.doi.org/10.1016/0167-7799(96)80930-9] [PMID: 8867291]
[41]
Hoffmann, F.; van den Heuvel, J.; Zidek, N.; Rinas, U. Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale. Enzyme Microb. Technol., 2004, 34(3-4), 235-241.
[http://dx.doi.org/10.1016/j.enzmictec.2003.10.011]
[42]
Marston, F.A.; Hartley, D.L. Solubilization of protein aggregates. Methods Enzymol., 1990, 182, 264-276.
[http://dx.doi.org/10.1016/0076-6879(90)82022-T] [PMID: 2179665]
[43]
Clark, E.D.B. Protein refolding for industrial processes. Curr. Opin. Biotechnol., 2001, 12(2), 202-207.
[http://dx.doi.org/10.1016/S0958-1669(00)00200-7] [PMID: 11287238]
[44]
Ahmadian, M.; Jahanian-Najafabadi, A.; Akbari, V. Optimization of buffer additives for efficient recovery of hGM-CSF from inclusion bodies using response surface methodology. Iran. J. Pharm. Res., 2020, 19(3), 297-309.
[PMID: 33680031]
[45]
Saxena, V.P.; Wetlaufer, D.B. Formation of three-dimensional structure in proteins. I. Rapid nonenzymic reactivation of reduced lysozyme. Biochemistry, 1970, 9(25), 5015-5023.
[http://dx.doi.org/10.1021/bi00827a028] [PMID: 4991411]
[46]
Dasari, V.K.R.; Are, D.; Joginapally, V.R.; Mangamoori, L.N.; Adibhatla, K.S.B.R. Optimization of the downstream process for high recovery of rhG-CSF from inclusion bodies expressed in Escherichia coli. Process Biochem., 2008, 43(5), 566-575.
[http://dx.doi.org/10.1016/j.procbio.2008.01.024]
[47]
Codevilla, C.F.; Brum, L., Jr; de Oliveira, P.R.; Dolman, C.; Rafferty, B.; Dalmora, S.L. Validation of an SEC‐HPLC method for the analysis of rhG‐CSF in pharmaceutical formulations. J. Liq. Chromatogr. Relat. Technol., 2004, 27(17), 2689-2698.
[http://dx.doi.org/10.1081/JLC-200029121]
[48]
Zhang, C.; Yang, X.L.; Yuan, Y.H.; Pu, J.; Liao, F. Site-specific PEGylation of therapeutic proteins via optimization of both accessible reactive amino acid residues and PEG derivatives. BioDrugs, 2012, 26(4), 209-215.
[http://dx.doi.org/10.1007/BF03261880] [PMID: 22721556]
[49]
Li, X-Q.; Lei, J-D.; Su, Z-G.; Ma, G-H. Comparison of bioactivities of monopegylated rhG-CSF with branched and linear mPEG. Process Biochem., 2007, 42(12), 1625-1631.
[http://dx.doi.org/10.1016/j.procbio.2007.09.005]
[50]
Veronese, F.M.; Mero, A.; Caboi, F.; Sergi, M.; Marongiu, C.; Pasut, G. Site-specific pegylation of G-CSF by reversible denaturation. Bioconjug. Chem., 2007, 18(6), 1824-1830.
[http://dx.doi.org/10.1021/bc070123+] [PMID: 17941684]
[51]
Kinstler, O.; Molineux, G.; Treuheit, M.; Ladd, D.; Gegg, C. Mono-N-terminal poly(ethylene glycol)-protein conjugates. Adv. Drug Deliv. Rev., 2002, 54(4), 477-485.
[http://dx.doi.org/10.1016/S0169-409X(02)00023-6] [PMID: 12052710]
[52]
Piedmonte, D.M.; Treuheit, M.J. Formulation of Neulasta (pegfilgrastim). Adv. Drug Deliv. Rev., 2008, 60(1), 50-58.
[http://dx.doi.org/10.1016/j.addr.2007.04.017] [PMID: 17822802]
[53]
Phillips, J.; Ritter, S.; Starner, C.; Gleason, P. Filgrastim (Neupogen) and pegfilgrastim (Neulasta): Cost analysis and utilization management opportunity assessment. J. Manag. Care Pharm., 2012, 18, 176-177.
[54]
Yun, Q.; Yang, R.E.; Chen, T.; Bi, J.; Ma, G.; Su, Z. Reproducible preparation and effective separation of PEGylated recombinant human granulocyte colony-stimulating factor with novel “PEG-pellet” PEGylation mode and ion-exchange chromatography. J. Biotechnol., 2005, 118(1), 67-74.
[http://dx.doi.org/10.1016/j.jbiotec.2005.02.015] [PMID: 15904987]
[55]
Pabst, T.M.; Buckley, J.J.; Ramasubramanyan, N.; Hunter, A.K. Comparison of strong anion-exchangers for the purification of a PEGylated protein. J. Chromatogr. A, 2007, 1147(2), 172-182.
[http://dx.doi.org/10.1016/j.chroma.2007.02.051] [PMID: 17346720]
[56]
Wang, J.; Hu, T.; Liu, Y.; Zhang, G.; Ma, G.; Su, Z. Kinetic and stoichiometric analysis of the modification process for N-terminal PEGylation of staphylokinase. Anal. Biochem., 2011, 412(1), 114-116.
[http://dx.doi.org/10.1016/j.ab.2010.12.030] [PMID: 21185800]
[57]
Werle, M.; Bernkop-Schnürch, A. Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids, 2006, 30(4), 351-367.
[http://dx.doi.org/10.1007/s00726-005-0289-3] [PMID: 16622600]
[58]
Lee, H.; Jang, I.H.; Ryu, S.H.; Park, T.G. N-terminal site-specific mono-PEGylation of epidermal growth factor. Pharm. Res., 2003, 20(5), 818-825.
[http://dx.doi.org/10.1023/A:1023402123119] [PMID: 12751640]
[59]
Na, D.H.; Lee, K.C.; DeLuca, P.P. PEGylation of octreotide: II. Effect of N-terminal mono-PEGylation on biological activity and pharmacokinetics. Pharm. Res., 2005, 22(5), 743-749.
[http://dx.doi.org/10.1007/s11095-005-2590-y] [PMID: 15906169]
[60]
Dou, H.; Zhang, M.; Zhang, Y.; Yin, C. Synthesis and purification of mono-PEGylated insulin. Chem. Biol. Drug Des., 2007, 69(2), 132-138.
[http://dx.doi.org/10.1111/j.1747-0285.2007.00479.x] [PMID: 17381727]
[61]
Mayolo-Deloisa, K.; Lienqueo, M.E.; Andrews, B.; Rito-Palomares, M.; Asenjo, J.A. Hydrophobic interaction chromatography for purification of monoPEGylated RNase A. J. Chromatogr. A, 2012, 1242, 11-16.
[http://dx.doi.org/10.1016/j.chroma.2012.03.079] [PMID: 22579360]
[62]
Yu, D.; Ghosh, R. Purification of PEGylated protein using membrane chromatography. J. Pharm. Sci., 2010, 99(8), 3326-3333.
[http://dx.doi.org/10.1002/jps.22103] [PMID: 20186937]
[63]
Khalilzadeh, R.; Mohammadian-Mosaabadi, J.; Bahrami, A.; Nazak-Tabbar, A.; Nasiri-Khalili, M.A.; Amouheidari, A. Process development for production of human granulocyte-colony stimulating factor by high cell density cultivation of recombinant Escherichia coli. J. Ind. Microbiol. Biotechnol., 2008, 35(12), 1643-1650.
[http://dx.doi.org/10.1007/s10295-008-0408-8] [PMID: 18682998]
[64]
Babaeipour, V.; Abbas, M.P.H.; Sahebnazar, Z.; Alizadeh, R. Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation. Bioprocess Biosyst. Eng., 2010, 33(5), 591-598.
[http://dx.doi.org/10.1007/s00449-009-0380-3] [PMID: 19859744]
[65]
Babaeipour, V.; Khanchezar, S.; Mofid, M.R.; Pesaran Hagi Abbas, M. Efficient process development of recombinant human granulocyte colony-stimulating factor (rh-GCSF) production in Escherichia coli. Iran. Biomed. J., 2015, 19(2), 102-110.
[PMID: 25864815]
[66]
Babaeipour, V.; Mofid, M.R.; Khanchezar, S.; Faraji, F.; Abolghasemi, S. Bench-scale Overproduction and Purification of recombinant GCSF in Escherichia coli fed-batch process. J. Appl. Pharm. Sci., 2017, 7(8), 149-155.
[67]
Ramya, M.; Selvarajan, E. Purification of human recombinant granulocyte colony stimulating factor from Escherichia coli. Afr. J. Biotechnol., 2012, 11(50), 11104-11109.
[http://dx.doi.org/10.5897/AJB12.133]
[68]
Rao, D.V.K.; Narasu, M.L.; Rao, A.K.S.B. A purification method for improving the process yield and quality of recombinant human granulocyte colony-stimulating factor expressed in Escherichia coli and its characterization. Biotechnol. Appl. Biochem., 2008, 50(Pt 2), 77-87.
[http://dx.doi.org/10.1042/BA20070130] [PMID: 17714071]
[69]
Peternel, S.; Komel, R. Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells. Microb. Cell Fact., 2010, 9(1), 66.
[http://dx.doi.org/10.1186/1475-2859-9-66] [PMID: 20831775]

Rights & Permissions Print Cite
Article Metrics
24
1
© 2024 Bentham Science Publishers | Privacy Policy