Biopolym. Cell. 2003; 19(6):530-535.
Molecular and Cell Biotechnologies
Influence of various glycerol concentrations on the yield and solubility of the Escherichia coli recombinant methionine aminopeptidase
1, 2Slavchenko I. Yu., 1Boreyko E. V., 1Vorobey N. V.
  1. PSRS "Biotechnolog"
    150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680
  2. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680


The influence of glycerol on the yield and solubility of a recombinant protein in a model system of E. coli methionine aminopeptidase (MAP) overproduction using the bacteriophage 77 RNA polymerase has been investigated. We have found, that glycerol added in a rich medium may inhibit the producer growth, promote the MAP accumulation in a soluble form and influence the target product yield. It has been shown, that manipulation of such parameters as glycerol concentration and culture optical density, when glycerol is added to the medium, allows purposely to increase the MAP yield in a soluble form. The approaches developed may be applicable for the production of other recombinant proteins in E. coli cells.


[1] Middelberg AP. Preparative protein refolding. Trends Biotechnol. 2002;20(10):437-43.
[2] Misawa S, Kumagai I. Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies. Biopolymers. 1999;51(4):297-307.
[3] Mukhopadhyay A. Inclusion bodies and purification of proteins in biologically active forms. Adv Biochem Eng Biotechnol. 1997;56:61-109.
[4] Schlieker C, Bukau B, Mogk A. Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology. J Biotechnol. 2002;96(1):13-21.
[5] Chao YP, Chiang CJ, Lo TE, Fu H. Overproduction of D-hydantoinase and carbamoylase in a soluble form in Escherichia coli. Appl Microbiol Biotechnol. 2000;54(3):348-53.
[6] Amrein KE, Takacs B, Stieger M, Molnos J, Flint NA, Burn P. Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL. Proc Natl Acad Sci U S A. 1995;92(4):1048-52.
[7] Shin CS, Hong MS, Kim DY, Shin HC, Lee J. Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli. Appl Microbiol Biotechnol. 1998;49(4):364-70.
[8] Thomas JG, Baneyx F. Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli. Protein Expr Purif. 1997;11(3):289-96.
[9] Nygaard FB, Harlow KW. Heterologous expression of soluble, active proteins in Escherichia coli: the human estrogen receptor hormone-binding domain as paradigm. Protein Expr Purif. 2001;21(3):500-9.
[10] Sawyer JR, Schlom J, Kashmiri SV. The effects of induction conditions on production of a soluble anti-tumor sFv in Escherichia coli. Protein Eng. 1994;7(11):1401-6.
[11] Georgiou G, Valax P, Ostermeier M, Horowitz PM. Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli. Protein Sci. 1994;3(11):1953-60.
[12] Bowden GA, Georgiou G. Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli. J Biol Chem. 1990;265(28):16760-6.
[13] Barth S, Huhn M, Matthey B, Klimka A, Galinski EA, Engert A. Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions. Appl Environ Microbiol. 2000;66(4):1572-9.
[14] Blackwell JR, Horgan R. A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Lett. 1991;295(1-3):10-2.
[15] Moore JT, Uppal A, Maley F, Maley GF. Overcoming inclusion body formation in a high-level expression system. Protein Expr Purif. 1993;4(2):160-3.
[16] Schein CH, Noteborn MHM. Formation of Soluble Recombinant Proteins in Escherichia Coli is Favored by Lower Growth Temperature. Bio/Technology. 1988;6(3):291–4.
[17] Slavchenko IYu, Boreyko EV, Gavrysh TG, Kostyuchenko IP, Kordyum VA. Biosynthesis of human basic fibroblast growth factor in soluble form in Escherichia coli and its purification. Biopolym Cell. 2003; 19(2):179-184.
[18] Slavchenko IYu, Boreyko EV, Vorobey NV, Gavrysh TG, Pehota EN, Kordyum VA. Overexpression and purification of methionine aminopeptidase from Escherichia coli. Biopolym Cell. 2003; 19(3):274-280.
[19] Slavchenko IYu, Boreyko EV, Vorobey NV, Chernykh SI, Kordyum VA. Overproduction of human α-2b interferon in a soluble form in Escherichia coli cells using the bacteriophage T7 RNA polymerase-base expression system. Biopolym Cell. 2003; 19(4):367-373.
[20] Sugimoto S, Yokoo Y, Hatakeyama N, Yotsuji A, Teshiba S, Hagino H. Higher culture pH is preferable for inclusion body formation of recombinant salmon growth hormone inEsherichia coli. Biotechnol Lett. 1991;13(6):385–8.
[21] Yang QH, Wu CL, Lin K, Li L. Low concentration of inducer favors production of active form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli. Protein Expr Purif. 1997;10(3):320-4.
[22] Park SJ, Gunsalus RP. Oxygen, iron, carbon, and superoxide control of the fumarase fumA and fumC genes of Escherichia coli: role of the arcA, fnr, and soxR gene products. J Bacteriol. 1995;177(21):6255-62.
[23] Kagawa N, Cao Q. Osmotic stress induced by carbohydrates enhances expression of foreign proteins in Escherichia coli. Arch Biochem Biophys. 2001;393(2):290-6.
[24] Leandro P, Lechner MC, Tavares de Almeida I, Konecki D. Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes produced in a prokaryotic expression system. Mol Genet Metab. 2001;73(2):173-8.
[25] Slavchenko IYu. The influence of organic carbon sources on the production of a recombinant protein in Escherichia coli cells. Biopolym Cell. 2002; 18(4):334-339.
[26] Hengge-Aronis R. Back to log phase: sigma S as a global regulator in the osmotic control of gene expression in Escherichia coli. Mol Microbiol. 1996;21(5):887-93.
[27] Diamant S, Eliahu N, Rosenthal D, Goloubinoff P. Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol Chem. 2001;276(43):39586-91.
[28] Eppler T, Boos W. Glycerol-3-phosphate-mediated repression of malT in Escherichia coli does not require metabolism, depends on enzyme IIAGlc and is mediated by cAMP levels. Mol Microbiol. 1999;33(6):1221-31.
[29] Fang A, Demain AL. Influence of aeration and carbon source on production of microcin B17 by Escherichia coli ZK650. Appl Microbiol Biotechnol. 1997;47(5):547-53.