Application Note 14

Efficient Uniform Labeling of Proteins Expressed in Baculovirus-Infected Insect Cells Using BioExpress^® 2000 (Insect Cell) Medium

André Strauss, Gabriele Fendrich, and Wolfgang Jahnke

Novartis Institutes for Biomedical Research, Basel, Switzerland

Uniform isotope labeling is a key tool for NMR studies on recombinant proteins and their interaction with ligands of pharmaceutical interest. For this purpose, most recombinant proteins have been expressed in labeled form using E. coli. However, such expression is restricted to proteins of a noncomplex nature. As expression of more complex proteins is routinely done in cell cultures of higher eukaryotes using mammalian or insect cells, the need for isotope-labeling methods for these expression systems is evident. 

The recent availability of a suitable labeling medium, BioExpress^® 2000 (insect cells) from CIL makes it possible to efficiently isotope label more complex proteins, such as kinases expressed in baculovirus (BV)-infected insect cells (Strauss, et al., 2005). BioExpress 2000 insect cell medium is commercially available from CIL in three different forms:
 • CGM-2000-U – unlabeled (BioExpress 2000-U)
 • CGM-2000-N – ¹⁵N-labeled (BioExpress 2000-N)
 • CGM-2000-CN – ¹³C/¹⁵N-labeled (BioExpress 2000-CN) 

The catalytic domain of Abl kinase (Abl) was used as model protein in this study, because:
 • Abl is a typical tyrosine kinase
 • Abl is well expressed in BV-infected insect cells
 • information on the crystal structure and on amino acid-specific isotope labeling is known1
 • Abl is a therapeutically relevant target

Successful uniform isotope labeling of proteins with BV-infected insect cells is dependent on: 
 • good expression of soluble and correctly folded protein in the labeling medium
 • a suitable labeling medium giving sufficiently high label incorporation
 • a suitable culture method compatible with labeling

To be useful for most important NMR studies, a labeled protein has to be expressed with BV-infected insect cells in the labeling medium in sufficient quality and quantity for isolation of at least 10 mg correctly folded protein. This can be easily achieved for Abl kinase with a half-liter of labeling medium. Upon resuspension of cells and expression in BioExpress 2000, Abl is the most abundant protein under these culture conditions as shown by SDS-PAGE analysis (Figure 1A). Expression levels were as high as 115 mg/L total Abl protein (Table 1) and 50-80 mg/L of isolated protein (Table 2). Similarly high expression under these conditions was also observed in BioExpress 2000 for another kinase (KDR) and two other proteins expressed in the BV system (Figure 1A), as well as several other proteins tested for expression (data not shown). For all these proteins, expression levels and cellular yield in expression cultures of Sf9 cells resuspended in BioExpress 2000 were similar to those achieved using standard expression media such as SF900 II (Invitrogen) or EX-CELL 420 (JRH) (Figure 1A, Table 1). For obtaining these and the following results on expression of Abl, 16 µM STI571 (a specific Abl kinase inhibitor) was added to the culture at infection in order to reduce phosphorylation and stabilize the protein.² 

Apart from allowing good expression of a recombinant protein, an isotope-labeling medium for BV-infected insect cells suitable for advanced NMR studies has to contain all 20 amino acids isotopically labeled at a high percentage to ensure high label incorporation into the recombinant protein. This is the case for both, the ¹⁵N-labeling medium (BioExpress 2000-N) and the ¹³C/¹⁵N-labeling medium (BioExpress 2000-CN), where high incorporation rates of 91.4% and 90.5%, respectively, have been found for Abl kinase expressed in BV-infected Sf9 cells (Table 2).²

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