A general outline for baculovirus expression is listed below. For one project, it usually takes approximately 2 months to accomplish these goals:
-Target gene will be cloned to a bacterial transfer vector with enhancer in polyhedrin promoter. The inserts are typically flanked by portions of viral genes to permit homologous recombination with replication defective, linear, viral DNA. Vectors or inserts may include 6 x his-tag sequences for protein targeting and purification. Direction of the inserts relative to the polyhedrin promoter will be verified and plasmid for co-transfection will be purified.
-Recombinant viruses are formed after intracellular homologous recombination between the ends of the viral molecules and portions of the transfer vector that flank the gene of interest. These recombination events insert your gene into the virus and complement defective viral gene(s) to permit viral replication. Non-recombinant viruses are kept to a minimal within this system.
-Transfected cell supernatants are harvested. Insect cells with dilutions of this supernatant are infected to isolate single virus plaque. Viral plaques in 3 ml infection are identified and can also be detected via Western blots.
-Additional insect cells are infected with viruses from these plaques to amplify the high titer of viral stocks. Protein expression in these cells may be examined in Western blots.
-The level of protein expression (MOI, time course of infection) is optimized and its activity is tested in the appropriate bioassay.
It is common to obtain high protein yield from E. coli expressions system. However, problems with proper folding and lack of post-translational processing may produce nonfunctional recombinant proteins. The Baculovirus Expression Vector System can be used to prevent, improve, and alleviate these problems. Heterologous gene expression in baculovirus systems offers several advantages over E. coli systems. Meanwhile, these viruses are considered safe for laboratory personnel. Insect cells infected with the virus recognize and properly process a variety of recognition signals in mammalian, plant and yeast genes, and provide moderate to high yields of biologically active proteins for study, diagnostic analysis and the other purposes etc. For the past twenty years in baculovirus development, scientists have not only been able to easily and conveniently express target protein in BEVS, but also developed various methods (serum free medium, insect cell lines and baculovirus virus genome etc.) to improve its efficiency.
In today’s world, more scientists are working on baculovirus expression systems for functional recombinant proteins and more people are discovering more complicated procedures to generate recombinant virus and produce recombinant proteins. However, it is very expensive to train technicians and set up a laboratory suitable for baculovirus expression. Thus, outsourcing is an alternative way to produce recombinant protein for current projects. Not only will you save money, but most importantly, you will also save time.
To understand outsourcing of protein project, the following should be considered:
Is it worth outsourcing to the BEVS laboratory? How much does a project cost? What is the typical yield of recombinant protein from an one liter expression? When will the outsourcing project be finished? Who is working on this project?
Here at Sheatech, Inc., we provide excellent outsourcing Baculovirus Expression Services. Our scientists, the department of R & D in Sheatech, Inc., have discovered several factors, which aid in the improvement of protein expression in Baculovirus Expression System. Not only can high quality baculovirus virus genome DNA generate high titer recombinant virus, but the enhancer in polyhedrin promoter has also provided and has been proven to show an increase in protein expression for most recombinant proteins. Both mid log insect cell culture as well as optimization conditions of infection have been considered to produce successful virus formation and protein recovery.