Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy.
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.
Xu
Zhi-Nan
ZN
Institute Bioengineering, School of Material Science and Chemical Engineering, Zhejiang University, Hangzhou 310027, China. znxu@zju.edu.cn
Shen
Wen-He
WH
Chen
Hao
H
Cen
Pei-Lin
PL
D000085:Acetates / Q000494:pharmacology; D002244:Carbon / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry* / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects* / Q000235:genetics*; D015316:Genetic Therapy / Q000295:instrumentation*; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D009584:Nitrogen / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D010957:Plasmids / Q000096:biosynthesis* / Q000235:genetics
D000085:Acetates; D003470:Culture Media; D010710:Phosphates; D002244:Carbon; D004247:DNA; D009584:Nitrogen