Optimization of Cultural Condition to Improve Recombinant C-D Fragment of Bacteriorhodopsin Production in E.coli

Document Type : Research Article

Authors

Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University, P.O. Box 14115-114 Tehran, I.R. IRAN

Abstract

At present study, a C-D fragment of bacteriorhodopsin (BR) in recombinant E. coli was expressed. BR mutant gene was synthesized by consideration of E. coli codon usage. The synthesized gene was cloned in pET21a+ expression plasmid at Nde I and Hind III restriction sites and expressed under T7 promoter successfully. The expressed protein was analyzed by SDS-PAGE. The effect of temperature (A), induction time (B) and the process time after induction (C), on the protein expression yield (the amount of product per unit dry cell mass) were screened using Yates table analysis. Three factors of A, B and C were significant and optimized by Taguchi method. The optimized condition was as: temperature 37 0C, induction time at OD600= 0.7 and the process time after induction 4 h. Also, the predicted protein production at optimized condition was 21.54% of total protein. Using the gained optimum condition, the effect of amino acid addition on expression level of recombinant C-D fragment of BR and bacterial growth in E.coli, using M9 culture medium was also investigated. Yates table analysis showed that Threonine, Leucine and Alanine are more effective and so were selected for addition to the culture medium in the fermentor, at two different levels. The results showed that amino acid addition caused increasing in the desired protein productivity, but had little effect on the bacterial growth.  

Keywords

Main Subjects

References

[1] Jong H.C., Ki C.K., Sang Y.L., Production of Recombinant Proteins by High Cell Density Culture of Escherichia coli, Chem Eng Sci., 61, p.876 (2006).
[2] Baneyx F., Recombinant Protein Expression in E.coli, Curr. Opin. Biotechnol., 10, p.411 (1999).
[3] Morten L.C., Niels T.E., Growth and Proton Exchange in Recombinant Escherichia coli BL21, Enzyme Microb. Technol., 31, p. 566 (2002).
[4] Jin Y., Girshevitz O., Friedman N., Ron I., Cahen D., Sheves M., Covalent Attachment of Bacteriorhodopsin Monolyer to Bromo-terminated Solid Supports: Preparation, Characterization, and Protein Stability, Chem Asian J., 3, p.1146 (2008).
[5] Luneburg J., Widmann M., Dathe M., Marti T., Secondary Structure of Bacteriorhodopsin Fragments, J. Biol. Chem., 273, p.28822 (1998).
[6] Nekrasova O.V., Wulfson A.N., Tikhonov R.V., Yakimov S.A., Simonova T.N., Tagvey A.I., Dolgikh D.A., Ostrovsky M.A., Kirpichnikov M.P., A New Hybrid Protein for Production of Recombinant Bacteriorhodopsin in Escherichia Coli, J. Biotechnol., 147, p.145 (2010).
[7] Xu J., Bhattacharya P., Varo G., Monolithically Integrated Bacterio- rhodopsin/semi-conductor Opto-electronic Integrated Circuit for a Bio-photoreceiver,  Biosens Bioelectron., 19, p.885 (2004).
[8] Pompejus M., Friedrich K., Teufel M., Fritz H.J., High-Yield Production of Bacteriorhodopsin via Expression of a Synthetic Gene in Escherichia coli, Eur. J. Biochem., 211, p. 27 (1993).
[9] Lee S.Y., Chang H.N., Um Y.S., Hong S.H., Bacteriorhodopsin Production by Cell Recycle Culture of Halobacterium Halobium, Biotechnol Lett., 20, p.763 (1998).
[10] Xu J., Stickrath A.B., Bhattacharya P., Nees J., Varo G., Hillebrecht J.R., Ren L., Birge R.R., Direct Measurement of the Photoelectric Response Time of Bacteriorhodopsin via Electro-Optic Sampling, Biophys. J., 85, p.1128 (2003).
[11] Walter J., Greenfield D., Liphardt J., Potential of Light-Harvesting Proton Pumps for Bioenergy Application, Curr. Opin. Biotechnol., 21, p.265 (2010).
[12] Oren A., Industrial and Environmental Applications of Halophilic Microorganisms, Environ. Technol., 31, p.825 (2010).
[13] Marti T., Refolding of Bacteriorhodopsin from Expressed Polypeptide Fragments, J. Biol. Chem., 273, p.9312 (1998).
[14] Santosh O., Ramchuran O.H., Eva, N.K., Effect of Postinduction Nutrient Feed Composition and Use of Lactose as Inducer During Production of  Thermostable Xylanase in E.scherichia coli Glucose-limited Fed-batch Cultivations, J. Biosci. Bioeng., 99, p.477 (2005).
[15] Yee L., Blanch H.W., Defined Media Optimization for Growth of Recombinant Escherichia coli, Biotechnol Bioeng., 41, p.221 (1993).
[16] Khalilzadeh R., Shojaosadati S.A., Bahrami A., Maghsoudi N.,Fed-batch Cultivation of Recombinant Escherichia coli Producing Human Interferon-γ under Controlled Specific Growth Rate, Iran J Biotechnol., 2, p.113 (2004).
[17] Shojaosadati S.A., Varedi Kolaei S.M., Babaeipour V., Farnoud A.M., Recent Advances in High Cell Density Cultivation for Production of Recombinant Protein, Iran. J. Biotechnol., 6, p.63 (2008).
[18] Sandén A.M., Prytz I., Tubulekas I., Förberg C., Le H., Hektor A., Neubauer P., Pragai Z., Harwood C., Ward A., Picon A., De Mattos J.T., Postma P., Farewell A., Nyström T., Reeh S., Pedersen S., Larsson G., Limiting Factors in Escherichia coli Fed-Batch Production ofRecombinant Proteins, Biotech Bioeng., 81, p. 158 (2003).

Files

Share

How to cite

Statistics