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Abstract: Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

[1] Cheng, C., Huang, Y.L., Yang, S.T., 1997. A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fed-batch fermentation. Biotechnol. Bioeng., 56:23-31.

[2] Churgay, L.M., Kovacevic, S., Tinsley, F.C., Kussow, C.M., Millican, R.L., Miller, J.R., 1997. Purification and characterization of secrected human leptin in baculovirus-infected insect cells. Gene, 190:131-137.

[3] DoHHs, S., 1996. Elimination of establishment license application for specified biotechnology and specified synthetic biological products. Notice Fed. Reg., 61:24227-24233.

[4] Durland, R.H., Eastman, E.M., 1998. Manufacturing and quality control of plasmid-based gene expression systems. Advanced Drug Delivery Reviews, 30:33-48.

[5] Ebisu, S., Takagi, H., Kadowaki, K., Yamagata, H., Udka, S., 1992. Production of human epidermal growth factor by Bacillus brevis increased with use of a stable plasmid from B. Brevis 481. Biosci. Biotechnol. Biochem., 56:812-813.

[6] Kim, B.G., Shuler, M.L., 1990. Analysis of pBR322 replication kinetics and its dependency on growth rate. Biotechnol. Bioeng., 36:233-242.

[7] Kleinman, M.J., Gingold, E.B., Stanbury, P.E., 1986. The stability of the yeast plasmid pJDB248 depends on the growth rate of the culture. Biotechnol. Lett., 8:225-230.

[8] Konstantinov, K., Kishimoto, M., Seki, T., Yoshida, T., 1990. A balanced Do-stat and application to the control of acetic acid excrection by recombinant E. coli. Biotechnol. Bioeng., 36:750-758.

[9] Lahijani, R., Hulley, G., Soriano, G., Horn, N.A., Marquet, M., 1996. High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperure-controllable point mutation. Hum. Gene Ther., 7:1971-1980.

[10] Li, X., Taylor, K.B., 1994. Effect of glucose on expression parameters of recombinant protein in E. coli during batch growth in complex medium. Biotechnol. Prog., 10:160-164.

[11] Naka, K., Risxhke, M., Sato, S., Markl, H., 1997. Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor. Appl. Microbiol. Biotechnol., 48:597-601.

[12] O’Kennedy, R.D., Baldwin, C., Keshavarz-Mmoore, E., 2000. Effects of growth medium selection on plasmid DNA production and initial processing steps. J Biotechnol., 76:175-183.

[13] Robbins, J.W., Taylor, K.B., 1989. Optimization of Escherichia-coli growth by controlled addition of glucose. Biotechnol. Bioeng., 34:1289-1294.

[14] SivaKesava, S., Xu, Z.N., Chen, Y., Hackett, J., Huang, R., Lam, E., Lam, T., Siu, K., Wong, R., Wong, W., 1999. Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system. Proc. Biochem., 34(9):893-900.

[15] Sun, W., Lee, C., George, H.A., 1993. Acetate inhibition on growth of recombinant E. coli and expression of fusion protein. Biotechnol. Lett., 15:809-814.

[16] Xu, Z., Liu, G., Cen, P., Wong, W.K.R., 2000. Factors influencing secretive production of human epidermal growth factor (hEGF) with recombinant E. coli K12. Bioproc. Eng., 23:669-674.

[17] Xu, Z.N., Chen, H., Tian, J., Cen, P.L., 2002. Studies on Cultivation Conditions for Efficient Production of Gene-therapy Vectors in E. coli. Proceeding of the Tenth NCBE, Hangzhou, China, p.43-48.