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Protein Expression
4.0 Insect Cell Expression System
4.1 Advantages
4.2 Disadvantages
4.3 Insect cell culture
4.4 Outline process of expression
4.5 Development of Vector and Host
4.0 Insect Cell Expression System
Insect cells are a higher eukaryotic system than yeast and are able to carry out more complex post-translational modifications than the other two systems (see Comparison of Expression Systems). They also have the best machinery for the folding of mammalian proteins and, therefore, give you the best chance of obtaining soluble protein when you want to express a protein of mammalian origin. The most commonly used vector system for recombinant protein expression in insect is baculovirus, although baculoviral also can be used for gene transfer and expression in mammalian cells. (Top)
4.1 Advantages
Baculovirus-assisted insect cell expression is optimal for glycosylated protein expression in a cost-effective manner. There are many advantages to using baculovirus for heterologous gene expression. Heterologous cDNA is expressed well. Proper transcriptional processing of genes with introns occurs but is expressed less efficiently. As with other eukaryotic expression systems, baculovirus expression of heterologous genes permits folding, post-translational modification and oligomerization in manners that are often identical to those that occur in mammalian cells. The insect cytoplasmic environment allows proper folding and S-S bond formation, unlike the reducing environment of the E. coli cytoplasm. Post-translational processing identical to that of mammalian cells has been reported for many proteins. These include proper proteolysis, N- and O-glycosylation, acylation, amidation, carboxymethylation, phosphorylation, and prenylation. Proteins may be secreted from cells or targeted to different subcellular locations. Single polypeptide, dimeric and trimeric proteins have been expressed in baculoviruses. Finally, expression of heterologous proteins is under the control of the strong polyhedrin promoter, allowing levels of expression of up to 30% of the total cell protein. (Top)
The benefits of protein expression with baculovirus can be summarized as:
Eukaryotic post-translational modification
Proper protein folding and function
High expression levels
Easy scale up with high-density suspension culture
Safety
Baculoviruses infect primarily insects with a narrow host range. Autographa californica, the most commonly used baculovirus for protein expression, infects only 2 lepidopteran (moth) families in nature. Although these viruses may enter other cells types (perhaps by phagocytosis), they are not infectious in them. For example, nucleocapsid proteins are not removed in most human cells. In human hepatic cell lines that do remove these proteins, the virus fails to replicate and express proteins due to the absence of insect transcription factors. Thus, working with baculoviruses is considered safe for humans and contamination of mammalian cell lines in shared biosafety hoods is not a problem. Recombinant DNA guidelines recommend a BL1 biosafety level for most baculovirus expression experiments. (Top)
4.2 Disadvantages
Despite these potential advantages, particular patterns of post-translational processing and expression must be empirically determined for each construct. Differences in proteins expressed by mammalian and baculovirus infected insect cells have been described and overcome in some cases. For example, inefficient secretion from insect cells may be circumvented by the addition of insect secretion signals (ex. honeybee melittin sequence). Improperly folded proteins and proteins that occur as intracellular aggregates may be due to expression late in the infection cycle. In such cases, harvesting cells at earlier times after infection may help. Low levels of expression can often be increased with optimization of time of expression and multiplicity of infection. The complete analysis of carbohydrate structures has been reported for a limited number of glycoproteins. Potential N-linked glycosylation sites are often either fully glycosylated or not glycosylated at all, as opposed to expression of various glycoforms that may occur in mammalian cells. Species-specific or tissue-specific modifications are unlikely to occur. (Top)
4.3 Insect cell culture
SF-9, SF-21, and High-Five insect cells are commonly used for baculovirus expression. SF-9 and SF-21 are ovarian cell lines from Spodoptera frugiperda. They are grown in Grace's (or a similar) media supplemented with 10% fetal calf serum, lactalbumin, and yeastolate. High-Five cells are egg cells from Trichoplusia ni. These cells are less expensive to maintain since they may be grown without fetal calf serum. They reportedly express higher levels of recombinant proteins, although we have found these differences to be minimal. All three cells lines may be grown at room temperature (optimum = 25 - 27°C), and do not require CO2 incubators. Their doubling time is 18-24 hours. (Top)
4.4 Outline process of expression
Given the variations of proteins to be expressed, no single protocol will satisfy every need. An outline for baculovirus expression is given below. One should plan 1-2 months to accomplish these goals:
  1. Ligate the gene of interest to a bacterial transfer vector. The inserts are typically flanked by portions of viral genes to permit homologous recombination with replication defective, linear, viral DNA. Vectors or inserts may include sequences for protein targeting and purification. Verify direction of the inserts relative to the polyhedrin promoter and purify plasmids for transfections into insect cells.
  1. Co-transfect insect cells with the recombinant transfer vector and a linearized viral vector. Electroporation, lipid, and calcium phosphate-mediated transformations work well. Replicative viruses are formed after intracellular homologous recombination between the ends of the viral molecules and portions of the transfer vector that flank the gene of interest. These recombination events insert your gene into the virus and complement defective viral gene(s) to permit viral replication. In some vectors, this may also generate production of marker proteins such as beta-galactosidase. Non-recombinant viruses are kept to a minimum with this system.
  1. Harvest the transfected cell supernatants.
  1. Infect insect cells with dilutions of this supernatant to isolate single virus plaques. Identification of viral plaques can be difficult without the presence of a marker gene product such as beta-galactosidase.
  1. 5) Infect additional insect cells with viruses from these plaques to amplify the quantity and titer of viral stocks. Protein expression in these cells may be examined in Western blots.
  1. Optimize the level of protein expression (MOI, time course of infection) and test for activity in an appropriate bioassay. (Top)
4.5 Development of Vector and Host
Baculoviral vector system and insect host cell lines are continually being developed. Several new systems for more robust and convenient application of baculovirus-based protein expression in insect cells have been invented and made available to researchers:
Transient expression system using non-viral vector system.
Multiple plasmid vector system for high-level expression of transgene in insect cells.
Novel insect cell lines with "humanized glycosylation function" for better glycosylation of expressed recombinant protein.
For other systems of protein expression, please visit the pages:

Yeast Expression System
Mammalian Expression System (Top)
Protein Expression
1.0 Overview of Protein Expression
1.1 Protein expression systems
1.2 GenWay's multi-tiered platform
1.3 Comparison of expression systems
1.4 Bioinformatics and structure analyses
2.0 E. coli Expression System
2.1 Initial expression screening
2.2 Optimization of expression levels
2.3 Improving protein solubility
2.4 Improving protein stability
2.5 Decreasing protein toxicity
2.6 In vitro expression using E. coli extracts
2.7 Co-expression
2.8 Functional protein expression example
3.0 Yeast Expression System
3.1 Superior expression
3.2 High cell densities
3.3 Controllable process
3.4 Mammalian-like proteins
3.5 Generations of stability
3.6 Durability
3.7 Maximum Value
3.8 Expression example
4.0 Mammalian Expression System
4.1 Usage of Mammalian Expression
4.2 Overview of mammalian expression
4.3 Transgene sequence optimization
4.4 Membrane protein expression
4.5 Production of fusion proteins
4.6 GenWay's specialty
5.0 Summary

Protein Expression Wikipedia Protein Expression Wikipedia 1.0 Protein Expression Wikipedia 2.0 Protein Expression Wikipedia 3.0
Protein Expression Wikipedia 5.0 Protein Expression Wikipedia 6.0
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