| Oct
1, 2005 |
| By:
Katie
Loomis, Courtney
Rockwell, Keith
Yaeger, Robert
Novy |
| Pharmaceutical
Discovery |
|
The InsectDirect™ System requires only 48 hours for transient
expression screening in Spodoptera insect cells and can be scaled up to
produce miligram quantities of protein.
Introduction
Figure 1. Comparison of traditional
baculovirus and InsectDirect™ System methods.
|
Producing heterologous proteins in
baculovirus expression systems is well established; however, creating,
titering, and amplifying viral stocks make it a time-intensive approach.
The recently developed InsectDirect™ System provides a rapid alternative
to baculovirus systems (1) (Figure 1). Here, we compare target protein
expression levels in both systems. Several targets, including two heat
shock proteins (HSPA and HSPB), a protein phosphatase (PP), and a protein
kinase (PK) were cloned, expressed, and purified.
Experimental Conditions
The InsectDirect System combines
expression vectors, a transfection reagent, lysis reagents, and Ni-NTA
His•Bind® Resin for optimal protein expression and purification. The
pIEx™ expression vectors feature transcriptional elements from Autographa
californica nuclear polyhedrosis virus (AcNPV): the hr5 (homologous
region 5) enhancer and the ie1 (immediate early 1) promoter. These
elements use endogenous insect cell transcriptional machinery, avoiding
baculovirus infection and associated cytopathic effects. Liposome-based
Insect GeneJuice® Transfection Reagent is optimized for maximal
transfection efficiency in Spodoptera insect cells (i.e., Sf9 and
Sf21 cells), with minimal toxicity in serum-containing or serum-free
media. Insect PopCulture® Reagent is a detergent-based lysis solution
designed to be added directly to insect cell cultures. Benzonase®
Nuclease degrades all forms of DNA and RNA, removing viscosity due to
chromosomal DNA.
Figure 2. Comparison of target
protein expression levels in a baculovirus system and the
InsectDirect System. Suspension cultures (10-mL cultures, 1 x 106
cells/mL) were infected (baculovirus) or transfected (InsectDirect),
incubated, and lysed. After removing crude fraction samples,
fusion proteins were purified. Samples representing 15 µL crude
or purified fractions were analyzed by 4-20% SDS-PAGE. Fusion tag
differences account for the protein size differences between the
vBAC and pIEx samples.
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Open reading frames for targets were
amplified and cloned into a baculovirus transfer vector, pBAC-2cp Ek/LIC,
and an InsectDirect vector, pIEx™-7 Ek/LIC. Both vectors encode
N-terminal His•Tag fusion sequences. Plasmids were isolated and the
pBAC-2cp recombinants were cotransfected with BacVector®-3000 DNA into
Sf9 cells to create baculovirus derivatives (vBACs). Pure baculovirus
recombinants were isolated after two rounds of plaque purification and
used to infect TriEx™ Sf9 cells. The pIEx recombinants were transfected
into Sf9 cells as well. Cells were incubated, lysates prepared, and fusion
proteins from infection (vBAC-2cp) and transfection (pIEx-7) were purified
using Ni-NTA His•Bind Resin. For specific experimental conditions, see www.emdbiosciences.com/docs/docs
/LIT/240021E.pdf
(2). Samples of total culture lysate (crude) and purified proteins
(purified) were analyzed by 4-20% SDS-PAGE (Figure 2).
Results
For these targets, our system
produced comparable or higher quantities of purified target protein. For
example, InsectDirect produced more than 845 µg purified HSPB, and the
baculovirus method produced 651 µg (2).
Conclusions
The InsectDirect System uses a
transient transfection method that saves time and resources while
generating purified target protein quantities comparable to baculovirus
infection. It can be used in conjunction with baculovirus for rapid,
small-scale expression screening. These processing and purification steps
can also be automated for high-throughput applications (3).
References
(1) K. Loomis, et al., inNovations
16, 7–10 (2002).
(2) K. Loomis, et al., inNovations
21, 12–14 (2005).
(3) K. Loomis, et al., inNovations
19, 10-12 (2004).
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