Custom Baculovirus Plasmid Cloning without Compatible Insert

Description

  Baculovirus is a helper independent viral system that is used to express heterologous genes. Because of their ability to efficiently infect a wide range of hosts, and their capacity to generate high yields of gene targets, the Baculovirus expression vector system (BEVS) has showed considerable promise to establish long-term gene expression in most cells. Polyhedrin and p10 are considered non-essential genes when baculovirus is in culture, allowing the coding sequences to be replaced with a sequence for the target protein. Compared to bacterial expression systems, the post-translational processing and folding of recombinant proteins produced in insect cells more closely resembles mammalian processes, and the yields of functional protein are often much greater.

Several unique features of the baculovirus system make this virus very appealing for preventive or therapeutic gene transfer. These include:

• Generate large amounts of recombinant protein
• Can express genes from bacteria, viruses, plants and mammals at levels from 1-500mg/l
• Several post translational modifications possible; phosphorylation, glycosylation and acylation

By pairing this technology with our expertise in virus packaging, abm offers custom Baculovirus subcloning and virus production, allowing high level gene expression in insect cells.

An overview of abm’s custom Baculovirus service:
Step 1: Gene synthesis and/or subcloning

• Strategy design and codon optimization
• Gene synthesis or amplification/isolation of the gene of interest from a customer-supplied vector and subcloning it into a transfer vector with His tag
• Sequence confirmation of insert
• Estimated time: 2 weeks

Step 2: Virus generation and expression evaluation

• Transformation of gene of interest into E. coli with baculoviral DNA
• Culture of E. coli and purification using mini-prep to produce an expression bacmid (recombinant viral DNA)
• Transfection of sf9 insect cells with expression bacmid
• Plaque purification and viral titer assay
• Small-scale culture to test expression level
• Estimated time: 4-6 weeks

Optional: Protein purification

• Virus stock preparation and amplification
• Large-scale culture and infection of insect cells for protein expression
• Purification from 1 litre of infected insect cell using affinity column of Ni-NTA or other column
• Estimated time: 4-5 weeks