Optimisation and evaluation of a high-throughput mammalian protein expression system.
Transient transfection of mammalian cells with episomal vectors is a very useful method for producing high levels of recombinant proteins. Transient systems remove the need for the laborious and time-consuming process of creating stable cell lines. Here, we describe the optimisation and evaluation of a high-throughput transient expression system in HEK293-EBNA cells. The process was developed for the expression of 10 constructs simultaneously in deep-well plates and subsequent purification using 96-well plate affinity chromatography. This enabled multiple combinations of different constructs, vectors, and expression conditions to be studied in parallel.
Davies
Angela
A
AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, UK. angela.davies@astrazeneca.com
Greene
April
A
Lullau
Elke
E
Abbott
W Mark
WM
D000818:Animals; D025901:Carboxypeptidase B2 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006639:Histidine / Q000235:genetics; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020442:Poloxamer / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D014162:Transfection / Q000379:methods
C485182:Cyan Fluorescent Protein; D019309:Epstein-Barr Virus Nuclear Antigens; D011994:Recombinant Proteins; D020442:Poloxamer; D049452:Green Fluorescent Proteins; C033223:polyhistidine; D006639:Histidine; C413545:glutathione S-transferase T1; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017346:Protein Serine-Threonine Kinases; D025901:Carboxypeptidase B2