Production of recombinant protein using the HeLa S3-vaccinia virus expression system: bioreactor perfusion and effects of post-infection temperature.
Adaptation of the vaccinia virus expression system to HeLa S3 suspension bioreactor culture for the production of recombinant protein was conducted. Evaluation of hollow fiber perfusion of suspension culture demonstrated its potential for increased cell density prior to infection. The hollow fiber was also used for medium manipulations prior to infection. Two process parameters, multiplicity of infection (MOI) and temperature during the protein production phase, were evaluated to determine their effect on expression of the reporter protein, enhanced green fluorescent protein (EGFP). An MOI of 1.0 was sufficient for infection and led to the highest level of intracellular EGFP expression. Reducing the temperature to 34 degrees C during the protein production phase increased production of the protein two-fold compared to 37 degrees C in spinner flask culture. Scaling up the process to a 1.5-liter bioreactor with hollow fiber perfusion led to an overall production level of 9.9 microg EGFP/10(6) infected cells, or 27 mg EGFP per liter.
Biotechnology Unit, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
Nicole A
NA
Bleckwenn
William E
WE
Bentley
Joseph
J
Shiloach
D019149:Bioreactors; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D006367:HeLa Cells / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013696:Temperature; D013997:Time Factors; D014616:Vaccinia virus / Q000235:genetics*
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins