Recombinant expression of proteins and their complexes using the baculovirus/insect cell expression system (BEVS)
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The baculovirus expression vector system (BEVS) is a powerful eukaryotic method to produce proteins and protein complexes. BEVS uses a recombinant baculovirus carrying the heterologous genes of interest, to infect insect cell cultures provided as monolayers or suspension. BEVS has become particularly attractive for protein research as it combines the ability to produce difficult-to-express, eukaryotic proteins with high yields and often authentic processing (PTMs) with simple cultivation needs without particular safety precautions. Important technological advances have greatly improved upon the original procedures for the generation of recombinant baculoviruses which were time consuming and required specialised expertise. Streamlined and cost-effective operation procedures for baculovirus-based expression have become available, and the BEVS is now accessible also to non-specialist users. Within the Infrastructure, new technologies have been developed and implemented which enable efficient and rapid production of large multiprotein complexes with many subunits, in the quality and quantity required for high-resolution structural and functional studies.
The main steps for production in insect cells using the baculovirus expression system are insertingf the gene(s) of interest into a baculoviral transfer vector of choice, followed by isolation of the recombinant baculovirus, amplification of viral stocks and infection of exponentially growing insect cell cultures for protein production.
Construction of transfer vectors will be mostly performed in the user's laboratory or on site, depending on the complexity of projects and the expertise of users. Quality controlled reagents, standard operating procedures in form of stream-lined protocols and expert subervision are provided by the host infrastructure. Assistance in planning and design of the experiment isprovided upon request by the user. Generation of the recombinant viruses, expression screening and medium-scale production is performed at the host infrastructure. The procedure entails from several days (e.g. cloning at user's laboratory and basic expression screening) to two/three weeks (e.g. multigene cloning and complex expression at an Instruct centre). Users return to their home laboratories with reagents including constructs, viruses, cell pellets and, in many cases, with purified samples.