Transfected insect cells in suspension culture rapidly yield moderate quantities of recombinant proteins in protein-free culture medium.
Methodology to rapidly express milligram quantities of recombinant proteins through the Lipofectin-mediated transfection of insect cells in small-scale, protein-free suspension culture is presented. The transfection phase in suspension culture was first optimized using the green fluorescence protein coupled with FACs analysis to examine the effect of variables such as the transfection media, duration, and cell density on transfection efficiency and expression level. The recombinant protein production phase was optimized using secreted alkaline phosphatase (SEAP) as a reporter protein to evaluate the cell seeding density and harvest time. Using this method, 5 secreted, 2 intracellular, and 1 chimeric protein were expressed at levels ranging from 6 to 50 mg/L. Furthermore, the ability to purify over 2 mg of His(6)-tagged SEAP by immobilized metal affinity chromatography from 50 mL insect cell culture medium to greater than 95% purity was also demonstrated. This method is suitable for scale-up and high-throughput applications.
Farrell
Patrick
P
Pharmaceutical Production Research Facility, Faculty of Engineering, University of Calgary, 2500 University Drive NW, Calgary, Alta, Canada T2N 1N4. pjfarrel@ucalgary.ca
Iatrou
Kostas
K
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002846:Chromatography, Affinity; D016895:Culture Media, Serum-Free; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007313:Insecta / Q000166:cytology; D010714:Phosphatidylethanolamines / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection / Q000379:methods*
D016895:Culture Media, Serum-Free; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C020888:1,2-dielaidoylphosphatidylethanolamine; D000469:Alkaline Phosphatase