Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Carboxypeptidase B2 / biosynthesis
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Carboxypeptidase B2 / genetics
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Carboxypeptidase B2 / isolation & purification
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Cell Culture Techniques / methods
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Cell Line
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Cell Proliferation / drug effects
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Epstein-Barr Virus Nuclear Antigens / genetics
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Gene Expression / genetics*
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Genetic Vectors / genetics
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Glutathione Transferase / biosynthesis
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Glutathione Transferase / genetics
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Glutathione Transferase / isolation & purification
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Green Fluorescent Proteins / biosynthesis
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / isolation & purification
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Histidine / genetics
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Humans
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Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / biosynthesis
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Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / genetics
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Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / isolation & purification
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Poloxamer / pharmacology
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Protein Serine-Threonine Kinases / biosynthesis
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / isolation & purification
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Protein-Tyrosine Kinases / biosynthesis
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Protein-Tyrosine Kinases / genetics
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Protein-Tyrosine Kinases / isolation & purification
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Recombinant Proteins / biosynthesis*
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Recombinant Proteins / isolation & purification
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Reproducibility of Results
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Transfection / methods
Substances
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Cyan Fluorescent Protein
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Epstein-Barr Virus Nuclear Antigens
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Recombinant Proteins
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Poloxamer
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Green Fluorescent Proteins
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polyhistidine
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Histidine
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glutathione S-transferase T1
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Glutathione Transferase
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Protein-Tyrosine Kinases
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Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
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Protein Serine-Threonine Kinases
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Carboxypeptidase B2