Application Note 20

Effective Site-Specific Isotopic Labeling (¹⁵N/¹³C Glycine; ¹⁵N/¹³C Phenylalanine; ¹⁵N Tryptophan) Expression Optimization Using BioExpress^® 2000 Media

Kenneth C. Bonanno

Vertex Pharmaceuticals, Inc., 130 Waverly Street, Cambridge, MA 02139 USA

Recombinant genes have been expressed in a variety of cellular systems for decades to generate protein reagents that are the potential targets for new small-molecule drugs. As these targets become more complex, researchers have developed innovative methods to study the structure of these proteins and the interactions with potential drugs. Cambridge Isotope Laboratories, Inc.’s (CIL) BioExpress® 2000 media incorporates isotope-labeled amino acids into recombinant protein expressed in baculovirus-infected (BV) insect cells to assist NMR structural studies (Strauss, et al., 2006). To use this labeled media most efficiently, expression conditions must be optimized at a small scale prior to production. 

The goal of this study was to maximize the expression of a histidine-tagged, human serine/threonine kinase in the Spodoptera frugiperda (SF9) cell line with site-specific isotopic labeling (¹⁵N/¹³C-labeled glycine; ¹⁵N/¹³C-labeled phenylalanine; ¹⁵N-labeled tryptophan) in BioExpress 2000 media (CGM-2000-Custom). The target enzyme was expressed in BV-infected insect cells cultured in various small volume (2 mL) conditions. Expression was optimized by adapting a previously designed high-throughput small-scale expression protocol (Chambers, et al., 2004) and incorporating Design of Experiment (DOE, Fisher, RA, 1925). 

Three conditions most widely optimized for insect cell expression are: 

• multiplicity of infection (MOI) 
• harvest time (hours post-infection – HPI 
• cell density at infection (cells/mL) 

A matrix of these variables was explored in this experiment to determine the maximum protein yield, measured in mg/L. 

A Box-Behnken response surface model (Box & Behnken, 1960) was applied in the experimental design using three equally spaced factors (low, medium, high) for each of the three conditions (MOI, HPI, cell density) in order to study the quantitative response, protein yield. This model provides 13 conditions, including a center­point with replicates. DOE allows the exploration of many variables via a limited number of experimental conditions using a statistically significant model produced by the experimental data. (Chambers and Swalley, 2008).

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