Effect of 4% glycerol and low aeration on result of expression in Escherichia coli of Cin3 and three Venturia inaequalis EST’s recombinant proteins

Taha H. Al-Samarrai, William T. Jones, Dawn Harvey, Christopher A. Kirk, M. Templtone

The Plant and Food Research Institute of New Zealand Ltd., CRI, Auckland, New Zealand.
The Plant and Food Research Institute of New Zealand Ltd., CRI, Palmerston North, New Zealand.
The University of Samarrai, College of Medical Technology, Samarra, Iraq.

DOI: 10.4236/ajmb.2013.31001                

Abstract

The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell wall. A novel Venturia inaequalis 5704 (Cin3) and three expressed sequence tags (ESTs); 38, 6987, and 4010 are strongly up-regulated in the early stages of infection. The CIN3 and three ESTs using two vectors pMAL-c2 and pET 21 were expressed in Escherichia coli. Recombinant proteins expression, solubility and yields were analyzed. 38, 5704 (Cin3) and 6987 re- combinant proteins were expressed in soluble form and while 4010 was expressed in inclusion bodies. Re- solution on native-PAGE, the recombinant proteins; 38, 5704 (Cin3), 6987 were shown to be present in dimmer, tetramer and polymer. A method was de- veloped, consisting of induction of expression at va- rious temperatures, and using enriched broth with 4% glycerol together with slow shaking, led to a decrease in concentration of nascent polypeptide and production of soluble recombinant proteins of; 38, 5704 (Cin3), 6987 and 4010. Resolution on native- PAGE, the recombinant proteins were shown to be present as monomer.

Keywords

Venturia inaequalis; Expressed Sequence Tag (ESTs); Phytopathogenic Fungus; Appressorium; A Stroma

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	MacHardy, W.E. (1996) Apple scab: Biology, epidemicology and management. APS Press, St. Paul.
[2]	Kucheryava, N., Bowen, J.K., Sutherland, P.W., Connolly, J.J., Mesarich, C.H., Rikkerink, E.H.A., Kemen, E., Plummer, K.M., Hahn, M. and Templeton, M.D. (2008) Two novel Venturia inaequalis genes induced upon morphogenetic differentiation during infection and in vitro on cellophane. Fungal Genetics and Biology, 45, 1329-1339. doi:10.1016/j.fgb.2008.07.010
[3]	Sambrook, J., Fritsch E.F. and Maniatis, T. (1989) Molecular cloning: A laboratory manual. 2nd Edition, Cold Spring Harbor Press, New York, 47-59.
[4]	Parks, T.D., Howard, E.D., Wolpert, T.J., Arp, D.J. and Dougherty, W.G. (1995) Expression and purification of a recombinant tobacco etch virus NIa proteinase: Biochemical analyses of the full-lengthand a naturally occurring truncated proteinase form. Virology, 210, 194-201. doi:10.1006/viro.1995.1331
[5]	Jones, W.T., Harvey, D., Kirk, C., Rakonjac, J., Sun, X., Frearson, N. and Al-Samarrai, T.H. (2007) Novelpeptide tag for detection and purification of recombinant expressed proteins. Protein Expression and Purification, 53, 404-410. doi:10.1016/j.pep.2007.01.006
[6]	Al-Samarrai, T.H., Kirk, C.A., Jones, W.T., Harvey, D. and Sun, X.L. (2007) Expression in Escherichia coli and in vitro refolding of the plant transcription factor Arabidopsis thaliana RGL3. Protein Expression and Purification, 53, 289-292. doi:10.1016/j.pep.2007.01.008
[7]	Martinez, A., Knappskog, P.M., Olafsdottir, S., D?skeland, A.P., Eiken, H.G., Svebak, R.M., Bozzini, M., Apold, J. and Flatmark, T. (1995) Expression of recombinant human phenylalanine hydroxylase asfusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases, Isolation and characterization of the wild-type enzyme. Biochemical Journal, 306, 589-597.
[8]	Jacquet, A., Daminet, V., Haumont, M. L., Garcia, Chaudoir, S., Bollen, A. and Biemans, R. (1999) Expression of a recombinant Toxoplasma gondii ROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation. Protein Expression and Purification, 17, 392-400. doi:10.1006/prep.1999.1150
[9]	Kapust, R.B. and Waugh, D.S. (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Science, 8, 1668-1674. doi:10.1110/ps.8.8.1668
[10]	Stevens, R.C. (2000) Design of high-throughput methods of protein production for structural biology. Structure, 8, 177-185. doi:10.1016/S0969-2126(00)00193-3
[11]	Georgiou, G. and Valax, P. (1996) Expression of correctly folded proteins in Escherichia coli. Current Opinion in Biotechnology, 7, 190-197. doi:10.1016/S0958-1669(96)80012-7
[12]	S?rensen, H.P. and Mortensen, K.K. (2005) Advanced genetic strategies for recombinant expression in Escherichia coli. Journal of Biotechnology, 115, 113-128. doi:10.1016/j.jbiotec.2004.08.004
[13]	Schein, C.H. (1989) Production of soluble recombinant proteins in bacteria. Biotechnology, 7, 1141-1148.
[14]	Vasina, J.A. and Baneyx, F. (1996) Recombinant protein expression at low temperatures under the transcriptional control of the major Escherichia coli cold shock promoter cspA. Applied and Environmental Microbiology, 62, 1444-1447.
[15]	Vanzi, F., Madan, B. and Sharp, K. (1998) Effect of the protein denaturants urea and guanidinium on water structure: A structural and thermodynamic study. Journal of the American Chemical Society, 120, 10748-10753. doi:10.1021/ja981529n
[16]	Jess, A.V. and Baney, F. (1997) Expression of aggregation-prone recombinant proteins at low temperatures: A comparative study of the Escherichia coli cspA and tac promoter systems. Protein Expression and Purification, 9, 211-218. doi:10.1006/prep.1996.0678
[17]	Chesshyre, J.A. and Hipkiss, A.R. (1989) Low temperatures stabilize interferon α-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli. Applied Microbiology and Biotechnology, 31, 158-162. doi:10.1007/BF00262455
[18]	Jiang, W., Hou Y. and Inouye, M. (1997) CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone. The Journal of Biological Chemistry, 272, 196-202. doi:10.1074/jbc.272.1.196
[19]	Arnold, U.R. and Ulbrich-Hofmann, R. (2001) Proteolytic degradation of ribonuclease A in the pretransition region of thermally and urea-induced unfolding. European Journal of Biochemistry, 268, 93-97. doi:10.1046/j.1432-1327.2001.01849.x
[20]	Sachdev, D. and Chirgwin, J.M. (1998) Solubility of proteins isolated from inclusion bodies enhanced by fusion to maltose-binding protein or Thioredoxin. Protein Expression and Purification, 12, 122-132. doi:10.1006/prep.1997.0826
[21]	Speed, M.A., Wang, D.I.C. and King, J. (1996) Specific aggregation of partially folded polypeptide chains: The molecular basis of inclusion body composition. Nature Biotechnology, 14, 1283. doi:10.1038/nbt1096-1283
[22]	Emerick, A.W., Bertolani, B.L., Ben-Bassat, A., White T.J. and Konrad, M.W. (1984) Expression of a beta-lactamase preproinsulin fusion protein in Escherichia coli. BioTechnology, 2, 165-168. doi:10.1038/nbt0284-165
[23]	Jurgen, B., Lin, H.Y., Riemschneider, S., Scharf, C., Neubauer, P., Schmid, R., Hecker, M. and Schweder, T. (2000) Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations. Biotechnology and Bioengineering, 70, 217-224. doi:10.1002/1097-0290(20001020)70:2<217::AID-BIT11>3.0.CO;2-W
[24]	Lesley, S.A., Graziano, J., Cho, C.Y., Knuth, M.W. and Klock, H.E. (2002) Gene expression response to misfolded protein as a screen for soluble recombinant protein. Protein Engineering, 15, 153-160. doi:10.1093/protein/15.2.153
[25]	Hoffmann, F. and Rinas, U. (2000) Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basicfibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli. Biotechnology Progress, 16, 1000-1007. doi:10.1021/bp0000959
[26]	Andersen, K.B. and von Meyenburg, K. (1980) Aregrowth rates of Escherichia coli in batch cultures limited by respiration? Journal of Bacteriology, 144, 114-123.
[27]	Xiu-bol, D.U., Sun, Y., Lin, F., Zheng, K.-Y., Wang, K.-W., Lin T.-T., Liu J.-Q., Shen, J.-C. and Luo G.-M. (2007) Chemical chaperones increasing expression level of soluble single-chain Fv antibody (scFv2F3). Chemical Research in Chinese Universities, 23, 69-75. doi:10.1016/S1005-9040(07)60014-2
[28]	Hansen, R. and Eriksen, N.T. (2007) Activity of recombinant GST in Escherichia coli grown on glucose and glycerol. Process Biochemistry, 42, 1259-1263. doi:10.1016/j.procbio.2007.05.022