The insect expression system is a method to obtain recombinant viruses by transposing the expression component in the transfer vector to the baculovirus shuttle vector proliferated in E. coli, extracting the shuttle plasmid DNA to transfect the insect cells, and then obtaining the offspring virus. The method of infiltrating the virus supernatant into insect cells to obtain the expressed recombinant protein. Through reviewing relevant literature, this paper summarizes the common problems in the process of insect expression experiments and analyzes the possible reasons and solutions.

1. Can Insect Cell Protein Expression Systems Co-express Two Genes?

Answer: Insect cells can express multiple genes at the same time, the baculovirus capsid can accommodate larger insert fragments of exogenous DNA, and two plasmids can be constructed separately, and then transfected together with insect cells, the advantage of this way is that the expression level of two proteins can be optimized respectively, and the expression balance of two proteins can be controlled, compared to the way of co-expression of one plasmid the disadvantage is that the plasmid is constructed before the expression of the proteins. The disadvantage of this method is that before protein expression, plasmid construction, plasmid extraction, protein viral encapsulation and cost are doubled; another way of co-expression is to construct two target genes on the pFastBac Dual vector, so that both proteins can be expressed at the same time. pFastBac Dual vector has two promoters, the PH promoter and P10 promoter respectively, wherein the PH promoter has a higher efficiency of protein expression than the P10 promoter.

2. How does the Preservation Method of Insect Viruses Affect the Viruses?

Answer: Add 2% cell culture grade BSA or fetal bovine serum when preparing the virus, it helps to protect the virus, if you forget to add it, add it after harvesting the virus, put it at 4 degrees or minus 80 for storage, short-term use (usually about half a year) can be put in 4 degrees, long-term use is recommended to be stored in minus 80, and it is recommended to test the viral titer or do an expression test to prevent the viral titer from decreasing or becoming invalid. For long-term projects, amplification projects can also be done. For long-term projects, amplification projects can also consider the preparation of BIIC, liquid nitrogen preservation.

3. What Kind of Protein is More Suitable for Insect Expression System?

Answer: Kinases, HDAC (histone deacetylase), viral/insect-derived proteins, glycoproteins, cytokines, coat proteins, VLP, envelope proteins, membrane proteins. Some of these proteins belong to the more difficult to express proteins themselves and can be expressed using the insect system to try.

4. Insect Protein Expression is to Use Insects?

Answer: No, it is through the culture of insect cells to express proteins, commonly used insect expression host cell lines are Sf9, Sf21, Hi5, S2, there are also insects to express proteins, such as bombyx, which belongs to the infested insects, belongs to the system of the bombyx (BmNPV), bombyx expression of proteins can be expressed in large quantities, but the purification of the proteins after the bombyx expression of the proteins will be very difficult, and the culture environment and technology of our bombyx are also limited, so we can not use insect system to express proteins. culture environment and technology are also limited, so we do not recommend the bombyx to express the protein.

5. Will the Target Protein be Released into the Extracellular Space Together with the Virus After Expression?

Answer: Whether the target protein is released into the extracellular space is mainly determined by the nature of the protein itself and whether the signal peptide is added. If the target protein has a signal peptide added during the construction of the carrier, the protein will generally be released into the extracellular space; however, if the target protein belongs to the intracellular proteins, such as cytoskeletal proteins, nuclear proteins and cytoplasmic proteins, the protein will not be released into the extracellular space even with the addition of the signal peptide. Therefore, the release of the virus and the release of the target protein are two processes, the protein may be released to the extracellular with the virus, or it may remain in the intracellular.

6. Is the Target Gene Integrated into the Gene of the Insect Cell?

Answer: No, exogenous genes are generally not easily integrated into the host genome, and specific conditions are required. In the insect system, the baculovirus carries the target gene into the host, and utilizes the host environment for self-replication and gene expression, and the target gene is not integrated into the host genome.

7. For Insect Protein Expression, is There Any Requirement for the Size of the Target Protein?

Answer: Generally, protein sizes of 10 kd-100 kd are suitable for any expression system, and insect expression systems are preferred for large proteins of 110-250 Kd.

8. What is the Difference Between Soluble Proteins Obtained by Adding Co-solvent and Soluble Proteins Obtained by Optimizing Expression at Low Temperatures? Will the Co-solvent Affect the Protein Activity?

Answer: The co-solvent is a relatively mild surfactant, which is to turn insoluble protein into soluble protein. Soluble proteins obtained by low-temperature optimized expression are soluble proteins expressed by optimizing conditions.

Theoretically, co-solvents have a small effect on protein activity, but it is not certain that co-solvents do not affect protein activity, mainly depending on what the subsequent experiments are doing, the demand for protein activity, if you are worried about the effect of co-solvents on protein, you can change the system expression or try to optimize the conditions.

There are many factors affecting protein activity, from the expression system, colon activity＜insect＜lactation, the colon often expresses proteins that are not active, insect activity is not guaranteed, lactation is most of the active; from the protein point of view: some proteins are poorly soluble, hydrophilicity is relatively poor, in the expression of easy to precipitate, sedimentation, basically no activity. Some proteins expressed itself is not active, no matter whether add co-solvent or not, the protein will not be active, so whether to add co-solvent and protein has no activity, not a reciprocal relationship, but if you need to have active proteins, it is best to express the soluble proteins, and do not need to add co-solvent.

KMD Bioscience has a perfect insect expression and purification system. The insect cell protein expression system is suitable for recombinant expression of membrane proteins, and 4 times and below transmembrane proteins can be almost effectively expressed and purified in insect cells. Purification of membrane proteins is time-consuming and labor-intensive. KMD Bioscience can provide customized products of full-length membrane proteins with purity up to 80%. KMD Bioscience can provide customers with a complete set of protein expression solutions, free of charge for the sequence analysis of the genes provided by customers; at the same time, according to the codon preference of Sf9, Sf21, Hi-5 and S2 insect cells, free of charge for you to optimize the codon, so as to effectively improve the expression of recombinant proteins, to provide you with one-stop service.