EMD Biosciences Inc., Madison, WI 53719; 2Caliper Life ...Insoluble 118 11 1023 85 NusA λPPase KDa...
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TP95 High-Throughput Automation of Protein Solubility and Concentration Screening with Microfluidic Protein Analysis Nathan Spangler 1 , Lynn Jordan 2 and Anthony Grabski 1 1 EMD Biosciences Inc., Madison, WI 53719; 2 Caliper Life Sciences Inc., Hopkinton, MA Abstract: High-throughput (HT) structural and functional proteomics requires the development of new reagents and automated methods to streamline the steps necessary to convert gene sequences to purified proteins. Automation has been applied successfully to cloning, expression and crystallography processes; however, HT solubility screening and purification remain critical bottlenecks in the protein structure-function determination pipeline. Our work focuses on testing a solubility screening kit designed for automated robotic liquid handling workstations. Solubility screening of recombinant proteins is performed using a 96 well format and reagents that are added directly to bacterial culture media. The unique reagents significantly increase protein extraction efficiency and reduce sample viscosity to facilitate automated liquid handling steps. A filter plate separates insoluble inclusion bodies from soluble proteins, which are collected in a 96-well plate for analysis. Insoluble proteins can be eluted from the filter plate and collected separately following solubilization in 4% SDS. Quantification is performed using a microfluidic version of 1D SDS-PAGE. This method provides excellent resolution and sensitivity across a broad range of salt concentrations, buffer types, and additives. Sizing, concentration, and % purity data can be reported for all proteins, if desired. The combination of the solubility kit, automated liquid handler, and microfluidic verification allow rapid, quantitative HT assessment of soluble and insoluble protein fractions in recombinant bacterial cultures. Protein Characterization Using the LabChip 90 Robotic Liquid Handling Work Station Caliper Life Science’s Sciclone ALH 3000 and a High Volume Head (volumes up to 200uL) using disposable tips is the basis of the workstation. Caliper Life Science’s 200uL Automation Certified Pipette Tips (P/N 56362) are used for all liquid handling steps including resuspension, lysing and transferring lysate to the filter plate. A deck mounted self-centering Microplate Shaker facilitates the resuspension of the pellet. A Positive Pressure Filtration System performs the filtration steps for the collecting the soluble and insoluble protein fractions. A Gripper is used to move the filter plate between the collection plates for the soluble and insoluble protein fractions. RoboPop solubility screening protocol on the Sciclone ALH 3000 • Add the PopCulture® and Lysonase™ Bioprocessing solution to cultures in a 96-well plate, mix and incubate for 10 minutes. • Transfer 200 ul of each sample to the 96-well filter plate positioned over a 96-well collection plate. • Apply positive pressure to the filter plate and elute the soluble proteins. • Reposition filter plate over a fresh collection plate and add 200 ul of 4% SDS to each well; mix and incubate 10 minutes. • Apply pressure to the filter plate to elute the detergent-solubilized inclusion bodies. Target Protein Yield and Apparent Molecular Weight Conclusion Combining the RoboPop™ solubility screening kit with the Sciclone ALH 3000 Workstation and LabChip® 90 Electrophoresis System eliminates the bottleneck of processing and analysis expression samples manually. The high throughput and ease-of-use offered by the automated method described here enables rapid screening of host-vector combinations and expression conditions to facilitate recombinant protein production for structural or functional studies. References Grabski, A.C., Drott, and D., Mehler M. (2003) inNovations 16, 11–13. Grabski, A.C., Drott, D., and Luedki, R. (2003) American Biotechnology Laboratory 35, 34-38. NusA λPPase ug/ml % in Fraction ug/ml % in Fraction Soluble 978 89 178 15 Insoluble 118 11 1023 85 NusA λPPase KDa 82 75 kDa 26 26 LC90 SDS PAGE A) Electropherograms B) Gel Images C) Coomassie-Stained SDS PAGE Figure 2. Sample Preparation, Processing and Analysis. pET recombinants encoding His•Tag® fusions of NusA and λ PPase were expressed in E. coli BL21(DE3). Overnight growth and in- duction was at 30°C in media containing Overnight Express™ System 1 (EMD Biosciences). Culture sample replicates (1-ml each) were extracted and fractionated robotically according to the RoboPop Solubility Screening protocol (EMD Biosciences) on the Sci- clone ALH 3000 liquid handler (Caliper Life Sciences). Target protein yields in soluble and insoluble fractions were measured with the LabChip 90 Electrophoresis system (Caliper Life Sciences). See panels A and B, above, for examples of LC-90 data output. For comparison, coomassie-stained SDS PAGE examples are shown in panel C. (10 – 20% gels, 5ul extract loaded per lane). Fluorescence Intensity Soluble Insoluble <NusA <λPPase <NusA <λPPase 225 150 100 75 50 35 25 15 10 Soluble Insoluble 225 150 100 75 50 35 25 15 10 < NusA < λPPase < Detergent Soluble Insoluble NusA NusA < λ PPase < λ PPase Figure 1. Time (Min) Time (Min)
Transcript of EMD Biosciences Inc., Madison, WI 53719; 2Caliper Life ...Insoluble 118 11 1023 85 NusA λPPase KDa...
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TP95 High-Throughput Automation of Protein Solubility and Concentration Screening with Microfluidic Protein AnalysisNathan Spangler1, Lynn Jordan2 and Anthony Grabski1 1EMD Biosciences Inc., Madison, WI 53719; 2Caliper Life Sciences Inc., Hopkinton, MA
Abstract:High-throughput (HT) structural and functional proteomics requires the development of new reagents and automated methods to streamline the steps necessary to convert gene sequences to purified proteins. Automation has been applied successfully to cloning, expression and crystallography processes; however, HT solubility screening and purification remain critical bottlenecks in the protein structure-function determination pipeline. Our work focuses on testing a solubility screening kit designed for automated robotic liquid handling workstations. Solubility screening of recombinant proteins is performed using a 96 well format and reagents that are added directly to bacterial culture media. The unique reagents significantly increase protein extraction efficiency and reduce sample viscosity to facilitate automated liquid handling steps. A filter plate separates insoluble inclusion bodies from soluble proteins, which are collected in a 96-well plate for analysis. Insoluble proteins can be eluted from the filter plate and collected separately following solubilization in 4% SDS.
Quantification is performed using a microfluidic version of 1D SDS-PAGE. This method provides excellent resolution and sensitivity across a broad range of salt concentrations, buffer types, and additives. Sizing, concentration, and % purity data can be reported for all proteins, if desired. The combination of the solubility kit, automated liquid handler, and microfluidic verification allow rapid, quantitative HT assessment of soluble and insoluble protein fractions in recombinant bacterial cultures.
Protein Characterization Using the LabChip 90
Robotic Liquid Handling Work StationCaliper Life Science’s Sciclone ALH 3000 and a High Volume Head (volumes up to 200uL) using disposable tips is the basis of the workstation. Caliper Life Science’s 200uL Automation Certified Pipette Tips (P/N 56362) are used for all liquid handling steps including resuspension, lysing and transferring lysate to the filter plate. A deck mounted self-centering Microplate Shaker facilitates the resuspension of the pellet. A Positive Pressure Filtration System performs the filtration steps for the collecting the soluble and insoluble protein fractions. A Gripper is used to move the filter plate
between the collection plates for the soluble and insoluble protein fractions.
RoboPop solubility screening protocol on the Sciclone ALH 3000• Add the PopCulture® and Lysonase™ Bioprocessing solution to cultures in a 96-well plate, mix and
incubate for 10 minutes.
• Transfer 200 ul of each sample to the 96-well filter plate positioned over a 96-well collection plate.
• Apply positive pressure to the filter plate and elute the soluble proteins.
• Reposition filter plate over a fresh collection plate and add 200 ul of 4% SDS to each well; mix and incubate 10 minutes.
• Apply pressure to the filter plate to elute the detergent-solubilized inclusion bodies.
Target Protein Yield and Apparent Molecular Weight
ConclusionCombining the RoboPop™ solubility screening kit with the Sciclone ALH 3000 Workstation and LabChip® 90 Electrophoresis System eliminates the bottleneck of processing and analysis expression samples manually. The high throughput and ease-of-use offered by the automated method described here enables rapid screening of host-vector combinations and expression conditions to facilitate recombinant protein production for structural or functional studies.
ReferencesGrabski, A.C., Drott, and D., Mehler M. (2003) inNovations 16, 11–13.
Grabski, A.C., Drott, D., and Luedki, R. (2003) American Biotechnology Laboratory 35, 34-38.
NusA λPPaseug/ml % in Fraction ug/ml % in Fraction
Soluble 978 89 178 15Insoluble 118 11 1023 85
NusA λPPaseKDa8275
kDa2626
LC90SDS PAGE
A) Electropherograms
B) Gel Images
C) Coomassie-Stained SDS PAGE
Figure 2. Sample Preparation, Processing and Analysis.pET recombinants encoding His•Tag® fusions of NusA and λ PPase were expressed in E. coli BL21(DE3). Overnight growth and in-duction was at 30°C in media containing Overnight Express™ System 1 (EMD Biosciences). Culture sample replicates (1-ml each) were extracted and fractionated robotically according to the RoboPop Solubility Screening protocol (EMD Biosciences) on the Sci-clone ALH 3000 liquid handler (Caliper Life Sciences). Target protein yields in soluble and insoluble fractions were measured with the LabChip 90 Electrophoresis system (Caliper Life Sciences). See panels A and B, above, for examples of LC-90 data output. For comparison, coomassie-stained SDS PAGE examples are shown in panel C. (10 – 20% gels, 5ul extract loaded per lane).
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Soluble Insoluble
