gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Baculovirus Recombinant Protein Expression Vectors
The baculovirus vector system is widely used for the expression of recombinant proteins in cultured insect cells. It is one of the most versatile and powerful systems for eukaryotic expression of recombinant proteins. This system is particularly advantageous for large-scale preparation of proteins that require expression in eukaryotic host cells. Many eukaryotic proteins undergo posttranslational modifications that can only take place in eukaryotic cells (e.g. glycosylation), or they need an eukaryotic cellular milieu for proper folding (e.g. membrane proteins). In these cases, prokaryotic expression systems are often inadequate and the baculovirus expression system could be a good alternative.
Baculovirus is a double-stranded DNA virus that commonly infects insects, particularly members of the order Lepidoptera (moths, butterflies and skippers). The cloning vector in our baculovirus expression system, pBV, is optimized for use with the baculovirus shuttle vector (known as bacmid) derived from the baculovirus strain AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus), which has a 134 kb genome in its wildtype form.
The gene of interest is first cloned into the pBV vector under the control of a strong promoter. This entire expression cassette, along with a gentamicin resistance gene, is flanked by the Tn7 transposon terminal elements, Tn7L and Tn7R. This vector is then transformed into E. coli carrying the bacmid shuttle vector and a helper plasmid. The bacmid is essentially a very large plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking site inserted in the lacZ coding region. The helper plasmid expresses the Tn7 transposase. The transposase would then mediate the transposition of the region flanked by Tn7R and Tn7L on the pBV vector, which contains the expression cassette for the gene of interest and gentamicin resistance, into the attTn7 docking site of the bacmid. Colonies containing recombinant bacmids can be identified by gentamicin selection and blue/white screening (non-recombinant colonies are blue due to lacZ expression whereas recombinant colonies are white due to disruption of lacZ by transposon insertion). Purified bacmid DNA can then be used to transfect insect cells to generate live baculovirus, which can be used to produce the recombinant protein of interest.
The most commonly used cell line for expressing recombinant proteins from baculovirus vectors is Sf9. This clonal line was derived from ovarian tissue of Spodoptera frugiperda (fall armyworm). This cell line is adaptable to a variety of culture and media conditions, including suspension or monolater culture and serum-free media. Larvae and other Lepidoptera cell lines have also been used extensively, and there are some reports of baculovirus being an effective vector for mammalian cells.
For further information about this vector system, please refer to the papers below.
|J Virol. 67:4566-79 (1993)||Generation of recombinant baculovirus by site-specific transposon-mediated insertion|
|Meth. Mol Med. 13:213-35 (1998)||Generation of recombinant baculovirus DNA in E.coli using a baculovirus shuttle vector|
Our baculovirus recombinant protein expression vector system enables efficient production of recombinant proteins in insect cells. This system allows for expression of proteins with posttranslational processing characteristic of eukaryotic cells, and with good adaptability to large-scale applications.
Eukaryotic system: Insect cells carry out posttranslational processing of proteins similar to that of mammalian cells. Our system is thus particularly suitable for expressing mammalian and other eukaryotic proteins whose function requires proper post-translational processing not present in prokaryotic expression systems, such as covalent modifications or membrane targeting.
Strong expression and good solubility: In most cases, the protein of interest is highly expressed, soluble, and can be easily recovered from infected cells.
Ease of scale-up: In our system, baculovirus obtained from initial transfection of insect cells can be used to infect more cells to further amplify viral titer. Protein production with our system can therefore be reproducibly scaled up.
Suspension culture: Sf9 and other Lepidoptera cell lines grow well in suspension cultures, allowing for the production of recombinant proteins in large-scale bioreactors.
Safety: Baculovirus cannot replicate outside of insect cells and are nonpathogenic to mammals and plants. Thus our expression system can be used in insect cell lines under minimal biosafety conditions.
Technical complexity: Protein production using the baculovirus expression system requires multiple steps, including cloning the gene of interest into pBV, generating recombinant bacmid from pBV, and transfecting bacmid into insect cells. These procedures are technical demanding and time consuming relative to recombinant protein expression in bacterial systems. These demands can be alleviated by choosing our recombinant bacmid generation and baculovirus packaging services when ordering your vector.
PH promoter: AcMNPV polyhedrin promoter. It drives high-level expression of the gene encoding your recombinant protein.
ORF: The open reading frame of your gene of interest is placed here.
SV40 early pA: Simian virus 40 early polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
Tn7L: Tn7 transposon left terminal element. It is recognized by Tn7 transposase. DNA flanked by Tn7R and Tn7L can be transposed by Tn7 transposase into attTn7 docking sites.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
Tn7R: Tn7 transposon right terminal element. It is recognized by Tn7 transposase. DNA flanked by Tn7R and Tn7L can be transposed by Tn7 transposase into attTn7 docking sites.
Gentamicin: Gentamicin resistance gene. It allows for drug selection of E. coli carrying recombinant bacmids.