Efficient uniform isotope 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 non-complex 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-infected insect cells (Strauss et al., 2005). This labeling medium is commercially available from CIL in three different forms: “BioExpress^® 2000 (Insect Cell) (Unlabeled)” (CIL, #CGM-2000-U), called here BioExpress^® 2000-U; “BioExpress^® 2000 (Insect Cell) (¹⁵N-labeled)” (CIL, #CGM-2000-N), called here BioExpress^® 2000-N; “BioExpress^® 2000 (Insect Cell) (¹³C/¹⁵N-labeled)” (CIL, #CGM- 2000-CN), called here 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, is well expressed in Baculovirus-infected insect cells, information on the crystal structure and on amino acid-specific isotope labeling is known (Strauss et al., 2003) and furthermore it is a therapeutically relevant target.

Successful uniform isotope labeling of proteins with Baculovirusinfected insect cells is dependent on:
a.) good expression of soluble and correctly folded protein in the labeling medium.
b.) a suitable labeling medium giving sufficiently high label incorporation.
c.) a suitable culture method compatible with labeling.

To be useful for most important NMR studies, a labeled protein has to be expressed with Baculovirus-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 half a liter of labeling medium. Upon re-suspension 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 Baculovirus 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 re-suspended 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 (Strauss et al., 2005).

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 BVinfected Sf9 cells (Table 2; Strauss et al., 2005).

For ¹³C/¹⁵N-labeling of Abl in BioExpress 2000-CN^®, it was shown (Strauss et al., 2005) that the carbohydrates have to be present in ¹³C-labeled form to avoid label dilution. With ¹⁵N-labeled Abl and ¹³C/¹⁵N-labeled Abl, high quality ¹H-¹⁵N-HSQC spectra with identical resonance patterns were obtained upon NMR analysis. The majority of the 277 amino acids of GAMDP-Abl(229-500) show up as clear and defined resonances as shown at the example of the ¹H-¹⁵N-HSQC spectrum of ¹⁵N-labeled Abl (Figure 2). The same is true for the ¹H- ¹⁵N-HSQC spectrum of ¹³C/¹⁵N -labeled Abl (Strauss et al., 2005).

The most suitable culture protocol for uniform isotope labeling of a recombinant protein involves an initial growth phase for the insect cells in an unlabeled growth/expression medium and subsequently an expression phase where cells have been transferred by centrifugation to the labeling medium, prior to infection with the recombinant BV. This protocol allows much higher expression levels of the labeled protein (as shown in Figure 1B) compared to one with growth and expression in labeling medium without medium change.

A summarized version of the successful protocol for uniform isotope labeling of Abl described above is given in Figure 3. General methods for working with insect cells and Baculovirus are given in several textbooks (e.g. O’Reilly et al., 1994). A more detailed description of the experimental protocol on uniform isotope labeling of Abl in BVinfected insect cells is given elsewhere (Strauss, et al., 2005).

This uniform isotope labeling should also be applicable to other insect cell lines, as high expression of Abl in BioExpress^®-2000-U with Sf21 or H5 cells was also achieved.

In conclusion, the method described opens up the possibility of efficient uniform isotope labeling of more complex recombinant proteins expressed in Baculovirus-infected insect cells.

References:
Strauss, A., Bitsch, F., Cutting, B., Fendrich, F., Graff, P., Liebetanz, J., Zurini, M. and Jahnke, W. (2003) J Biomol NMR, 26, 367-372.

Strauss, A., Bitsch, F., Fendrich, F., Graff, P., Knecht, R., Meyhack, B. and Jahnke, W. (2005) J Biomol NMR, 31, 343-349.

O’Reilly, D.R., Miller, L.K. and Luckow, V.A. (1994) Baculovirus Expression Vectors, Oxford University Press, New York, NY

BioExpress^® is a registered trademark of Cambridge Isotope Laboratories

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