Sequential and simultaneous statistical optimization by dynamic design of experiment for peptide overexpression in recombinant Escherichia coli.
The production of recombinant anti-HIV peptide, T-20, in Escherichia coli was optimized by statistical experimental designs (successive designs with multifactors) such as 2(4-1) fractional factorial, 2(3) full factorial, and 2(2) rotational central composite design in order. The effects of media compositions (glucose, NPK sources, MgSO4, and trace elements), induction level, induction timing (optical density at induction process), and induction duration (culture time after induction) on T-20 production were studied by using a statistical response surface method. A series of iterative experimental designs was employed to determine optimal fermentation conditions (media and process factors). Optimal ranges characterized by %T-20 (proportion of peptide to the total cell protein) were observed, narrowed down, and further investigated to determine the optimal combination of culture conditions, which was as follows: 9, 6, 10, and 1 mL of glucose, NPK sources, MgSO4, and trace elements, respectively, in a total of 100 mL of medium inducted at an OD of 0.55-0.75 with 0.7 mM isopropyl-beta-D-thiogalactopyranoside in an induction duration of 4 h. Under these conditions, up to 14% of T-20 was obtained. This statistical optimization allowed the production of T-20 to be increased more than twofold (from 6 to 14%) within a shorter induction duration (from 6 to 4 h) at the shake-flask scale.
Lee
Kwang-Min
KM
Cellular Metabolic Engineering Lab., Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong Yuseong-gu Daejeon, 305-701, South Korea. Klee0922@yahoo.co.kr
Rhee
Chang-Hoon
CH
Kang
Choong-Kyung
CK
Kim
Jung-Hoe
JH
D019380:Anti-HIV Agents / Q000494:pharmacology*; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D003470:Culture Media; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D008278:Magnesium Sulfate / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D012044:Regression Analysis; D013997:Time Factors
D019380:Anti-HIV Agents; D003470:Culture Media; D010455:Peptides; D011994:Recombinant Proteins; D008278:Magnesium Sulfate