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J Biomol Tech. 2007 February; 18(1): 76.
PMCID: PMC2291865

P220-S Dual-Purpose Insect Cell Expression Vector for Transient Transfection and Baculovirus Generation

Abstract

Baculovirus-mediated expression has proven to be a robust method of generating recombinant proteins from insect cells. However, generating baculovirus recombinants using traditional techniques is time consuming and tedious. To accelerate the process of insect cell expression, EMD developed a rapid transient transfection-based approach, the InsectDirect System. This approach is well suited for the rapid generation of small to moderate amounts of recombinant protein. For situations that demand the baculovirus approach, we introduced a technology for rapid baculovirus production, BacMagic DNA. To optimize, simplify, and consolidate both systems. we created a single expression vector compatible with both approaches, pIEx/Bac.

To direct expression by transient transfection, pIEx/Bac expression vectors feature the homologous region 5 (hr5) enhancer and the immediate early 1 (ie1) promoter from Autographa californica nuclear polyhedrosis virus (AcNPV), a promoter/enhancer combination that uses endogenous insect-cell transcriptional machinery. To direct expression during baculovirus infection, the above promoter/enhancer combination is active during the early stage of baculovirus infection, and an additional p10 promoter directs expression in the late/very late phases. We also created additional vectors featuring alternative combinations of baculovirus-derived promoters and enhancers to verify that our particular combination of enhancer and promoter elements was optimal. Using the Radiance Ek/LIC cloning method, the reporter enzyme Renilla luciferase (Rluc) was cloned into all variants to allow comparisons of relative expression strength. The plasmid that gave the best overall performance in both systems was chosen as our new dual-purpose expression plasmid, pIEx/Bac. To further test the vector, we cloned additional inserts encoding differing classes of proteins, including an importin, a phosphatase, and kinases. The results demonstrate the utility of the pIEx/Bac vector for streamlined expression in insect cells.


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