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Gateway technology, the technology of constructing insect cell expression vector

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In order to improve the high-level and soluble expression of recombinant protein, a protein expression system can be constructed. The baculovirus vector expression system is an efficient eukaryotic expression system that can use baculovirus as a carrier for foreign genes to express foreign proteins by infecting insect cells. Gateway technology is a very convenient vector construction method, which can integrate the target gene into the target vector to obtain the recombinant plasmid of the protein expression vector. This technology greatly simplifies the steps of gene cloning and subcloning, while the typical cloning efficiency is high. When the gene shuttles between the target expression vectors quickly and easily, it can also ensure the correct direction and reading frame. Gateway technology also helps to purify and detect the expression of tagged proteins with different numbers.

Insect cells mainly produce simple N-glycans with terminal mannose residues, which will affect the overall structure and function of the glycoprotein of interest. Transgenic mammalian glycosylation genes are transferred into insect cells to establish transgenic insect cell lines, which can express complex glycosylation modified recombinant proteins. Insect cell baculovirus vector expression systems have become powerful tools for the expression of exogenous recombinant proteins. The construction and screening methods of recombinant insect systems mainly use the BaculoGold system established by linearization technology, the Bac-to-Bac system established by the Escherichia coli-insect cell shuttle vector technology, and the flashBAC system established on the basis of these two technologies. And the BaculoDirect system built with Gateway technology. There are also a variety of optimized systems constructed by using genetic engineering technology to knock out part of BV’s own genes or add multiple promoters. Gateway technology can transfer target genes to multiple expression systems, facilitating the analysis of functional genes and protein expression.

Insect cell vector expression system has the advantages of high safety, high expression level of recombinant protein, simultaneous expression of multiple genes, and complete post-translational modification of recombinant protein. It has been widely used in the fields of recombinant protein drugs, vaccines and antibodies, and gene therapy. Medicilon researchers have established a mature baculovirus-insect cell expression service platform, providing services including the preparation of recombinant baculovirus, the expression and purification of recombinant proteins and their complexes.

At present, the expression of kinase proteins is mostly achieved by insect cell expression systems. The BacMam expression system is an improvement of the traditional baculovirus expression system. Some researchers have used the BacMam system to express the receptor type tyrosine kinase human FGFR1, aiming to obtain more Recombinant protein close to natural structure and function [1]. First, the polyhedrin promoter of the pDEST20 vector was replaced with the CMV enhancer promoter to obtain the BacMam expression vector pDEST20-CMVe. Through the Gateway system, the region of FGFR1 with tyrosine kinase activity was cloned into the modified vector. Use insect cell Sf9 to produce virus, and P2 generation virus was transduced into mammalian cell FreeStyle 293-F for expression. After affinity purification, a 75 kD fusion protein was successfully obtained. The activity of this FGFR1 kinase was detected by the reaction of phosphorylated substrate. The results show that the BacMam system is a good platform for the expression of recombinant kinase FGFR1, and the kinase activity expressed is higher than that of the kinase protein expressed by insect cells.

The recombinant baculovirus system is a very useful tool for expressing recombinant proteins in insect cells. It is popular because of its easy folding, post-transcriptional modification, packaging, and high expression level. Currently, the preparation of insect cell expression vectors can combine Gateway technology with linearized parental virus DNA technology. Because there is no single expression system protein suitable for every protein, the best way to optimize gene expression is to analyze expression in multiple systems.

[1] BacMam system construction, expression, purification and identification of human FGFR1 [J]

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