High level of soluble expression and purification of catalytically active native UDP-galactose 4-epimerase of Aeromonas hydrophila in E. coli
Keshav Gopal, Shivani Agarwal, Aparna Dixit
DOI: 10.4236/abb.2011.26058   PDF   HTML     4,259 Downloads   7,897 Views  


The ubiquitous Aeromonas hydrophila is responsible for several pathological conditions in fish and human. Like most gram negative bacteria, its virulence relies on outer membrane lipopolysachharide (LPS). The Leloir pathway enzyme UDP-galactose 4-epimerase (GalE), plays an important role in the LPS biosynthesis, and therefore is a potential drug target. We have earlier carried out extensive biochemical and biophysical studies with histidine-tagged recombinant GalE. However, for effective drug design it is desirable to understand the structure-function relation of a protein in its native form without any additional sequences or tags. In the present study, we report the high level expression, purification and characterization of recombinant GalE (rGalE) of Aeromonas hydrophila in its native form in E coli. The rGalE expressed as a soluble protein was purified to near homogeneity. From 1 L of shake flask culture ~15 mg of purified rGalE was obtained. The purified protein was biologically active with Km and Kcat values of 0.7 mM and 28.8 s–1, respectively. The enzyme exhibited a temperature and pH optima of 37C and 7 - 9, respectively. Thus, the present study employed for soluble expression and purification of functionally active rGalE without any tag bypasses the need for cumbersome strategies associated with removal of tag from purified protein.

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Gopal, K. , Agarwal, S. and Dixit, A. (2011) High level of soluble expression and purification of catalytically active native UDP-galactose 4-epimerase of Aeromonas hydrophila in E. coli. Advances in Bioscience and Biotechnology, 2, 397-403. doi: 10.4236/abb.2011.26058.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Janda, J.M. (1991) Recent advances in the study of taxonomy, pathogenicity, and infectious syndromes associated with the genus Aeromonas. Clinical Microbiology Reviews, 4, 397-410.
[2] Kaper, J.B., Lockman, H., Colwell, R.R. and Joseph, S.W. (1980) Aeromonas hydrophila: ecology and toxigenicity of isolates form an estuary. Journal of Applied Microbiology, 50, 359-377. doi:10.1111/j.1365-2672.1981.tb00900.x
[3] Van der Kooj, D. (1988) Properties of Aeromonads and their occurrence and hygienic significance in drinking water. Zentralblatt Bakteriology Hygien B, 187, 1-17.
[4] Hazen, T.C., Flierman, C.B., Hirsch, R.P. and Esch, G.W. (1978) Prevalence and distribution of Aeromonas hydrophila in the United States. Applied and Environmental Microbiology, 36, 731-738.
[5] Janda, J.M. and Duffy, P.S. (1998) Mesophilic Aeromonads in human disease: Current taxonomy, laboratory identification, and infectious disease spectrum. Reviews of Infectious Diseases, 10, 980-997. doi:10.1093/clinids/10.5.980
[6] Pierson, D.E. and Carlson, S. (1996) Identification of the gale gene and a gale homolog and characterization of their roles in the biosynthesis of lipopolysaccharide in a serotype O: 8 strain of Yersinia enterocolitica. Journal of Bacteriology, 178, 5916-5924.
[7] Fry, B.N., Feng, S., Chen, Y.Y., Newell, D.G., Coloe, P.J. and Korolik V. (2000) The GalE gene of Campylobacter jejuni is involved in lipopolysaccharide synthesis and virulence. Analytical Biochemistry, 283, 64-70.
[8] Maxwell, E.S. (1957) The enzymic interconversion of uridine diphosphogalactose and uridine diphosphoglucose. Journal of Biological Chemistry, 229, 139-151.
[9] Vorgias, C.E., Lemaire, H.G. and Wilson, K.S. (1991) Over expression and purification of the galactose Operon enzymes from Escherichia coli. Protein Expression and Purifaction, 2, 330-338. doi:10.1016/1046-5928(91)90091-V
[10] Wilson, B.D. and Hogness, D.S. (1966) Galactokinase and Uridine diphosphogalactose 4-epimerase from Escherichia coli. Methods in Enzymology, VIII, Academic Press, New York, 229-240.
[11] Wilson, B.D. and Hogness, D.S. (1964) The enzymes of the galactose Operon in Escherichia coli I. Purification and characterization of uridine diphosphogalactose 4- epimerase. Journal of Biological Chemistry, 239, 2469- 2481.
[12] Houng, H.H., Kopecko, D.J. and Baron, L.S. (1990) Molecular cloning and physical and functional characterization of the Salmonella typhimurium and Salmonella typhi galactose utilization operons. Journal of Bacteriology, 172, 4392-4398.
[13] Potter, M.D. and Lo, R.Y. (1996) Cloning and characterization of the galE locus of Pasteurella haemolytica A1. Infection and Immunity, 64, 855-860.
[14] Stevenson, G. and Manning, P.A. (1985) Galactose epimeraseless (GalE) mutant G30 of Salmonella typhimurium is a good potential live oral vaccine carrier for fimbrial antigens. FEMS Microbiology Letters, 28, 317-321. doi:10.1111/j.1574-6968.1985.tb00813.x
[15] Fernández de Henestrosa, A.R., Badiola, I., Saco, M., Perez de Rozas, A.M., Campoy, S. and Barbé, J. (1997) Importance of the galE gene on the virulence of Pasteurella multocida. FEMS Microbiology Letters, 154, 311- 316. doi:10.1016/S0378-1097(97)00347-9
[16] Petrovska, L., Hewinson, R.G., Dougan, G., Maskell, D.J. and Woodward, M.J. (1999) Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant. Veterinary Microbiology, 65, 21-36. doi:10.1016/S0378-1135(98)00281-8
[17] Sun, Q.M., Chen, L.L., Cao, L., Fang, L., Chen, C. and Hua, Z.C. (2005) An improved strategy for high-level production of human vasostatin120-180. Biotechnology Progress, 21, 1048-1052. doi:10.1021/bp049583x
[18] Tang, W., Sun, Z.Y., Pannell, R., Gurewich, V. and Liu, J.N.(1997) An efficient system for production of recombinant urokinase-type plasminogen activator. Protein Expression and Purification, 11, 279-283. doi:10.1006/prep.1997.0800
[19] Busso, D., Kim, R. and Kim, S.H. (2003) Expression of soluble recombinant proteins in a cell-free system using a 96-well format. Journal of Biochemical and Biophysical Methods, 55, 233-240. doi:10.1016/S0165-022X(03)00049-6
[20] Amor-Mahjoub, M., Suppini, J.P., Gomez-Vrielyunck, N. and Ladjimi, M. (2006) The effect of the hexahistidine- tag in the oligomerization of HSC70 constructs. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 844, 328-334. doi:10.1016/j.jchromb.2006.07.031
[21] Bucher, M.H., Evdokimov, A.G. and Waugh, D.S. (2002) Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein. Acta Crystallographica D, 58, 392-397. doi:10.1107/S0907444901021187
[22] Cadel, S., Gouzy-Darmon, C., Petres, S., Piesse, C., Pham, V.L., Beinfeld, M.C., Cohen, P. and Foulon, T. (2004) Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells. Protein Expression and Purification, 36, 19-30. doi:10.1016/j.pep.2004.03.013
[23] Freydank, A.C., Brandt, W. and Dr?ger, B.(2008) Protein structure modeling indicates hexahistidine-tag interference with enzyme activity. Proteins, 72, 173-183. doi:10.1002/prot.21905
[24] Schmitt, J., Hess, H. and Stunnenberg, H.G. (1993) Affinity purification of histidine-tagged proteins. Molecular Biology Reports, 18, 223-230. doi:10.1007/BF01674434
[25] Jenny, R.J., Mann, K.G. and Lundblad, R.L. (2003) A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa. Protein Expression and Purification, 31, 1-11. doi:10.1016/S1046-5928(03)00168-2
[26] Agarwal, S., Gopal, K., Chhabra, G. and Dixit, A. (2009) Molecular cloning, sequence analysis and homology modeling of galE encoding UDP-galactose 4-epimerase of Aeromonas hydrophila. Bioinformation, 4, 216-222.
[27] Agarwal, S., Gopal, K., Upadhyaya, T. and Dixit, A. (2007) Biochemical and functional characterization of UDP-galactose 4-epimerase from Aeromonas hydrophila. Biochimica et Biophysica Acta, 1774, 828-837.
[28] Mathur, D. and Garg, L.C. (2007) Functional phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv: Rapid purification with high yield and purity. Protein Expression and Purification, 52, 373-378. doi:10.1016/j.pep.2006.10.006
[29] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.
[30] Smyth, D.R., Mrozkiewicz, M.K., McGrath, W.J., Listwan, P. and Kobe, B. (2003) Crystal structures of fusion proteins with large-affinity tags. Protein Science, 12, 1313-1322. doi:10.1110/ps.0243403
[31] Kenig, M., Peternel, S., Gaberc-Porekar, V. and Menart, V. (2006) Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins. Journal of Chromatography A, 1101, 293-306. doi:10.1016/j.chroma.2005.09.089
[32] Galloway, C.A., Sowden, M.P. and Smith, H.C. (2003) Increasing the yield of soluble recombinant protein expressed in E. coli by induction during late log phase. BioTechniques, 34, 524-530.
[33] Roper, J.R. and Ferguson Michael, A.J. (2003) Cloning and characterization of the UDP-glucose 4-epimerase of Trypanosoma cruzi. Molecular and Biochemical Parasitology, 132, 47-53. doi:10.1016/j.molbiopara.2003.07.002
[34] Dormann, P. and Benning, C. (1996) Functional expression of uridine 5’-diphosphoglucose 4-epimerase (EC from Arabidopsis thaliana in Saccharomyces cerevisiae and Escherichia coli. Archives of Biochemistry and Biophysics, 327, 27-34. doi:10.1006/abbi.1996.0088
[35] Berger, E., Arabshahi, A., Wei, Y., Schilling, J.F. and Frey, P.A. (2001) Acid-base catalysis by UDP-galactose 4-epimerase: correlations of kinetically measured acid dissociation constants with thermodynamic values for tyrosine 149. Biochemistry, 40, 6699-6705. doi:10.1021/bi0104571
[36] Combet, C., Blanchet, C., Geourjon, C. and Deléage, G.. (2000) NPS@: Network Protein Sequence Analysis. Trends in Biochemical Sciences, 25, 147-150. doi:10.1016/S0968-0004(99)01540-6

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