Secretory N-Linked Glycoprotein Expression approach

That the baculovirus expression system is unable to produce recombinant glycoprotein containing authentic N-glycan restrict the production of some therapeutic mammalian protein via insect cell. After years of hard work of researchers, this limitation had been addressed by either expressing additional glycozymes or construct transgenic cell line or combining of both strategy.

However, the above reforms are all based on the transformation of the host cell DNA itself, and only limited to cell lines like Sf9 and Sf21 which are suitable for non secretory protein expression.

For the secretory protein, cell line derived from Trichoplusia ni have been demonstrated to produce significantly higher amounts in many cases. Creative BioMart provides a N-Linked Glycoprotein Expression service via the approach in which the baculovirus gene is manipulated rather than insect cell. The approach is called SweetBac. The manipulated baculovirus DNA can be used to infect any host insect cell line theoretically, thus can be combined with the cell lines derived from Trichoplusia ni to produce secretory N-linked glycoprotein.

Choosing our Secretory N-linked Glycoprotein Expression Service, customers only need to provide the coding sequence of your target protein. We will provide customizes service from codon optimization to large-scale expression and purification. For more assistance, please contact us for help.

Technology Description of SweetBac expression approach

The secretory n-linked glycoprotein expression approach is based on the MultiBac expression approach which support the multi sequence insertion and simultaneous protein expression. All the modifications are carried out on the baculovirus DNA. A so-called glyco-module, which is consists of acetylglucosaminyltransferase, galactosyltransferase, polyhedrin and p10 is introduced into the baculovirus genome via the LoxP recombination site to form the novel virus DNA for the SweetBac approach.

Secretory N-Linked Glycoprotein Expression approach

Service Contents and Deliverables

Here are the service contents of secretory n-linked glycoprotein expression approach available at Creative BioMart.

Step		Service Description	Timeline	Deliverables
1	System Selection	Select donor vector Select acceptor vector Select competent E. coli cell	——	——
2	Generating vectors contains multigene expression cassettes	Multigene construction via homing endonuclease (HE) / BstXI multiplication Multigene construction using Cre-Lox recombination Combining HE / BstXI and Cre-Lox recombination	2-3 weeks	Multigene acceptor and donor vectors
3	Preparation of recombinant Bacmid	Cre – LoxP fusion of donor and acceptor vectors Transformation fusion vectors into E. coli Recombinant MultiBacmid preparation	1 week	Recombinant MultiBacmid Enzymatic / Sequencing validation report.
4	Recombinant Baculovirus Production	Transfect the insect cells with recombinant Bacmid Produce P1 Virus stock (low titer), P2 Virus stock (high titer), P1, P2 Western Blot and titer assay	2 weeks	Recombinant virus suspension WB report Titer test report
5	Small-Scale Expression & Purification	P2 virus transfect insect cell Protein purification SDS-PAGE, Western Blot	1.5 weeks	Target protein sample Quantitative test report
6	Optional Services	System optimization Secondary purification Tag removal Activity assay	1-2 weeks	Optimization solution Protein product Protein activity report
7	Large-Scale Expression & Purification (1-10L) (optional)	Culture amplification Western Blot Purification	1-3 weeks	Purified Protein QC report

Application of N- Linked Glycoprotein Expression Service

Production of targets for drug discovery and assay development
Antibody development against membrane proteins like GPCRs and ion channels
Manufacturing of biologics in insect cells with humanized glycosylation patterns.

Please note that all products/services provided are for research use only. Not intended for any clinical use.