Chapter 9 Expression Screening in Mammalian Suspension Cells Susan D. Chapple and Michael R. Dyson Abstract Proteins naturally expressed in eukaryotic organisms often require host chaperones, binding partners, and posttranslational modifications for correct folding. Ideally the heterologous expression system chosen should be as similar to the natural host as possible. For example, mammalian proteins should be expressed in mammalian expression systems. However this does not guarantee a protein will be expressed in a sufficient high yield for structural or biochemical studies or antibody generation. Often a screening process is undertaken in which many variants including truncations, point mutations, investigation of orthologues, fusion to peptide or protein tags at the N- or C-terminus, the co-expression of binding partners, and even culture conditions are varied to identify the optimal expression conditions. This requires multi-parallel expression screening in mammalian cells similar to that already described for E. coli expression. Here we describe in detail a multi-parallel method to express proteins in mammalian suspension cells by transient transfection in 24-well blocks. Key words Expression screening, HEK293 cells, CHO cells, Transient transfection, Mammalian cell culture, Interaction assays, Antibodies
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Introduction Expression of human and mammalian proteins in E. coli often results in a poor soluble expression yield [1]. Expression in eukaryotic systems such as yeast or insect cells can aid expression. However the most authentic chaperones, binding partners, and posttranslational modifications for mammalian proteins will be found in mammalian expression systems. There are several reasons why one may wish to perform a multi-parallel expression experiment. Firstly it is common to express single or tandem domains of multi-domain containing proteins to both improve expression and to study their function. Unfortunately domain boundaries are not accurately predicted within the current protein databases [2] and so often several truncations are performed at the DNA level either by rational or combinatorial [3] design followed by expression screening. Secondly individual expression domains can be stabilized and their
Yu Wai Chen (ed.), Structural Genomics: General Applications, Methods in Molecular Biology, vol. 1091, DOI 10.1007/978-1-62703-691-7_9, © Springer Science+Business Media, LLC 2014
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yield improved by fusion at the N- or C-terminus with peptide or protein tags [4]. Each protein target is different and so it is likely that several fusion partners would need to be investigated. Thirdly, it is well known that protein orthologues and mutations can display different solubility and crystallization properties and so one may wish to investigate a panel of point mutations and orthologues. Lastly some proteins are only stable in the presence of their natural binding partners and so one may wish to investigate coexpression with candidate binding partners [5]. The variables described here soon multiply and a thorough investigation requires the use of a plate based mammalian expression screen. The optimization of expression parameters is not the only reason an investigator may wish to perform a multi-parallel expression experiment. They may also, for example, need to express a panel of receptor ectodomains for interaction studies [6] or functional screening [7]. Also panels of recombinant antibodies can be expressed for screening in proteomic applications [8, 9] or to aid therapeutic antibody lead isolation and optimization projects [10]. Screening expression in suspension adapted HEK293 or CHO cells allows the convenience of fast scale-up of any hits discovered in a small-scale expression screen [11, 12]. Here we describe a method for transfection of HEK293F cells in 24-well blocks and a dot-blot screen to identify secreted expression screen hits. The dot blot screen could be replaced by a standard western blot procedure or ELISA. Also the methods are transferable to suspension Chinese Hamster Ovary (CHO) suspension cells.
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Materials All chemicals were from Sigma unless stated otherwise.
2.1 HEK293F Cell Maintenance
1. For maintenance of cells in Erlenmeyer flasks a humidified CO2 shake incubator is required with a 25 mm orbital throw such as the Infors Multitron. 2. Vented sterile Erlenmeyer flasks (Corning). 3. HEK293F cells and Freestyle media (Life Technologies). 4. A hemocytometer for cell counting.
2.2 HEK293F Cell 24-Well Block Transfection
1. For maintenance of cells in 24-well blocks a humidified CO2 plate shaker incubator is required with a 3 mm orbital throw such as the Infors Multitron plate shaker incubator. 2. Sterile 24-well blocks were from Qiagen (Fig. 1). 3. Linear 25 kDa polyethylenimine (PEI) was from Polysciences Inc. This was prepared at a concentration of 1 mg/ml in MilliQ water. Solubilization was achieved by first adding
Multiplexed Transient Transfection of HEK293 Cells
145
Fig. 1 24-well block for HEK293F cell culturing and transfection
concentrated HCl to a stirred PEI solution until the pH was
1
Introduction Expression of human and mammalian proteins in E. coli often results in a poor soluble expression yield [1]. Expression in eukaryotic systems such as yeast or insect cells can aid expression. However the most authentic chaperones, binding partners, and posttranslational modifications for mammalian proteins will be found in mammalian expression systems. There are several reasons why one may wish to perform a multi-parallel expression experiment. Firstly it is common to express single or tandem domains of multi-domain containing proteins to both improve expression and to study their function. Unfortunately domain boundaries are not accurately predicted within the current protein databases [2] and so often several truncations are performed at the DNA level either by rational or combinatorial [3] design followed by expression screening. Secondly individual expression domains can be stabilized and their
Yu Wai Chen (ed.), Structural Genomics: General Applications, Methods in Molecular Biology, vol. 1091, DOI 10.1007/978-1-62703-691-7_9, © Springer Science+Business Media, LLC 2014
143
144
Susan D. Chapple and Michael R. Dyson
yield improved by fusion at the N- or C-terminus with peptide or protein tags [4]. Each protein target is different and so it is likely that several fusion partners would need to be investigated. Thirdly, it is well known that protein orthologues and mutations can display different solubility and crystallization properties and so one may wish to investigate a panel of point mutations and orthologues. Lastly some proteins are only stable in the presence of their natural binding partners and so one may wish to investigate coexpression with candidate binding partners [5]. The variables described here soon multiply and a thorough investigation requires the use of a plate based mammalian expression screen. The optimization of expression parameters is not the only reason an investigator may wish to perform a multi-parallel expression experiment. They may also, for example, need to express a panel of receptor ectodomains for interaction studies [6] or functional screening [7]. Also panels of recombinant antibodies can be expressed for screening in proteomic applications [8, 9] or to aid therapeutic antibody lead isolation and optimization projects [10]. Screening expression in suspension adapted HEK293 or CHO cells allows the convenience of fast scale-up of any hits discovered in a small-scale expression screen [11, 12]. Here we describe a method for transfection of HEK293F cells in 24-well blocks and a dot-blot screen to identify secreted expression screen hits. The dot blot screen could be replaced by a standard western blot procedure or ELISA. Also the methods are transferable to suspension Chinese Hamster Ovary (CHO) suspension cells.
2
Materials All chemicals were from Sigma unless stated otherwise.
2.1 HEK293F Cell Maintenance
1. For maintenance of cells in Erlenmeyer flasks a humidified CO2 shake incubator is required with a 25 mm orbital throw such as the Infors Multitron. 2. Vented sterile Erlenmeyer flasks (Corning). 3. HEK293F cells and Freestyle media (Life Technologies). 4. A hemocytometer for cell counting.
2.2 HEK293F Cell 24-Well Block Transfection
1. For maintenance of cells in 24-well blocks a humidified CO2 plate shaker incubator is required with a 3 mm orbital throw such as the Infors Multitron plate shaker incubator. 2. Sterile 24-well blocks were from Qiagen (Fig. 1). 3. Linear 25 kDa polyethylenimine (PEI) was from Polysciences Inc. This was prepared at a concentration of 1 mg/ml in MilliQ water. Solubilization was achieved by first adding
Multiplexed Transient Transfection of HEK293 Cells
145
Fig. 1 24-well block for HEK293F cell culturing and transfection
concentrated HCl to a stirred PEI solution until the pH was
