baculovirus kinase

benniegeorge authored almost 4 years ago

Authors: Creative Biogene

Baculovirus expression vector system (BEVS) is one of the four major expression systems of genetic engineering which belongs to the eukaryotic expression system. BEVS is widely used to express recombinant protein kinase and protein complexes. Moreover, BEVS is recognized as a powerful tool for the expression of large-scale protein on the strength of convenient process, high expression level and the function of post-translational modifications.

Technical Protocol

(1) Subcloning/PCR Cloning Clone your gene of interest into a baculovirus transfer vector. Either a standard subcloning or a PCR cloning will be used to transfer the gene of interest into the vector. Clones will be screened for the correct restriction fragments and sequenced.

(2) Recombinant Virus Production Cotransfect the baculovirus transfer vector into Sf9, Sf21 or Hi-5 according to actual situation. The low -titer recombinant viruses will be amplified 2-3 times to generate high-titer virus stocks.

(3) Optimization of Expression Recombinant protein expression will be optimized in one or more cell lines by doing a time-course study of infection, varying the multiplicity of infection (MOI). At each time point, the expressed protein will be analyzed by SDS-PAGE and Western blot. The results of these experiments will be used to determine the optimal conditions for protein production.

(4) Protein Characterization Recombinant protein from a small-scale culture will be characterized for cellular location, solubility, and accessibility of purification tags. In addition, the yield per unit volume will be estimated. Using the results of these experiments, a strategy for producing and purifying the protein will be made.

(5) Protein Production and Purification Production of the recombinant protein can be scaled up between one and several liter volumes. You will receive the cell pellets or conditioned media and a complete project report with the SDS-PAGE and/or Western blot analysis that document protein production. Purify your recombinant proteins using affinity chromatography, ion-exchange and gel filtration chromatography. Large-scale purification is also available.

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