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1. High Expression Levels

Baculoviruses can produce large quantities of recombinant proteins, often yielding 50-1000 mg per liter of culture. This high productivity makes it an attractive system for large-scale protein production.

Post-Translational Modification (PTM)

Insect cells can perform many eukaryotic post-translational modifications, including:

N- and O-linked glycosylation

Phosphorylation

Acylation

Amidation

Carboxymethylation

Isoprenylation
(This capability allows for the production of proteins that closely resemble their native counterparts in higher eukaryotes.)

3. Large insert capacity:

Baculoviruses can accommodate large DNA inserts, enabling the expression of large proteins or multiple genes simultaneously. This is useful for producing protein complexes or studying protein-protein interactions.

4. Proper protein folding:

The environment within insect cells supports the proper folding of complex proteins, frequently leading to functionally active recombinant proteins. This is essential for producing enzymes, receptors, and other proteins that need specific tertiary structures to be active.

5. Safety:

Baculoviruses are non-pathogenic to humans and other vertebrates, making them safer to work with compared to mammalian viruses. This reduces biosafety concerns and simplifies laboratory procedures.

6. Scalability:

The system is easily scalable from small-scale laboratory production to large-scale industrial manufacturing. Insect cells can be grown in suspension culture, allowing for easy scale-up in bioreactors.

7. Cost-effectiveness:

Insect cell culture is generally less expensive than mammalian cell culture systems. The media requirements are simpler, and the cells can grow at room temperature without CO2 supplementation.

8. Rapid production:

Recombinant protein expression can be detected within 48-72 hours post-infection, allowing for quick turnaround times in protein production.

9. Versatility:

The system can be used to produce a wide range of proteins, including cytoplasmic, nuclear, membrane-bound, and secreted proteins.

10. Co-expression capabilities:

Multiple genes can be expressed simultaneously, facilitating the production of protein complexes or proteins requiring co-factors for proper folding or activity.

11. High-throughput potential:

The system is amenable to high throughput screening approaches, making it valuable for structural genomics and drug discovery projects.

12. Mammalian-like glycosylation:

While not identical to mammalian glycosylation, insect cells can produce proteins with glycosylation patterns more similar to mammalian cells than bacterial systems, which is important for many therapeutic proteins.

13. Reduced proteolytic degradations:

Insect cells generally have lower levels of endogenous proteases compared to some mammalian cell lines, potentially leading to higher yields of intact recombinant proteins.

14. Flexibility in promoter choice:

The system allows for the use of various promoters, including the strong polyhedrin and p10 promoters, enabling fine-tuning of expression levels.

15. Availability of optimized tools:

Numerous commercially available vectors, cell lines, and reagents have been optimized for the baculovirus system, simplifying its implementation in research and industrial settings.

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Eukaryotic Expression:

Baculoviruses infect insect cells, which are eukaryotic. This is crucial for expressing proteins that require post-translational modifications (PTMs) like glycosylation, phosphorylation, and proper folding, which prokaryotic systems (like bacteria) often cannot provide. These PTMs are essential for the proper function and activity of many eukaryotic proteins, especially human proteins.

High Expression Levels:

The baculovirus system can achieve very high levels of protein expression, often comparable to or even exceeding those of other eukaryotic systems. This is due to the strong viral promoters used to drive the expression of the target gene.

Large Insert Capacity:

Baculoviruses have a relatively large genome, allowing for the insertion of large genes or even multiple genes. This is advantageous for expressing complex proteins or for co-expressing multiple subunits of a protein complex.

Safety:

Baculoviruses are generally considered safe to work with because they have a narrow host range, primarily infecting insects. They do not infect vertebrates (including humans) or plants, making them suitable for producing biopharmaceuticals.

Scalability:

Insect cell cultures can be grown in large-scale bioreactors, making the baculovirus system suitable for large-scale protein production for research, industrial, or therapeutic purposes.

Versatility:

The baculovirus system can be used to express a wide range of proteins, including:

Cytoplasmic proteins

Secreted proteins

Membrane proteins

Viral proteins

Enzymes

Antibodies

Two Main Methods for Recombinant Virus Production:

Co-transfection: This traditional method involves co-transfecting insect cells with a transfer plasmid containing the target gene and a linearized baculovirus genome. Homologous recombination within the cells generates the recombinant baculovirus.

Bac-to-Bac system: This more modern and efficient method uses bacterial cells to generate recombinant baculoviruses. It involves a series of genetic manipulations in bacteria to create a bacmid (a bacterial plasmid containing the baculovirus genome) with the target gene. This bacmid is then transfected into insect cells to produce the recombinant virus.

References:

1. Baculovirus expression system. Baculovirus Expression System - an overview | ScienceDirect Topics. (n.d.). https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/baculovirus-expression-system#:~:text=In%20contrast%20to%20other%20commonly,infectious%20virus%20in%20mammalian%20cells.

2. Felberbaum, R. S. (2015, May). The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors. Biotechnology journal. https://pmc.ncbi.nlm.nih.gov/articles/PMC7159335/

3. Baculovirus expression system. Baculovirus Expression System - an overview | ScienceDirect Topics. (n.d.). https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/baculovirus-expression-system#:~:text=In%20contrast%20to%20other%20commonly,infectious%20virus%20in%20mammalian%20cells.

4. Overview of the baculovirus expression system. (n.d.-a). https://radar.brookes.ac.uk/radar/file/0bf34266-b697-4b0f-ba29-e3f61d74ba03/1/Unit%205.4%20Overview%20of%20the%20Baculovirus%20Expression%20System%20accepted%20version%20(1).pdf

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Limitation of Baculovirus Expression System

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Limitation of Baculovirus Expression System

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1. Transient Transgene Expression

Baculoviruses induce transient transgene expression in mammalian cells.

In vivo: Transgene expression typically declines by day 7 and disappears by day 14 to 21.

In vitro: Transgene expression using baculoviruses is significantly shorter compared to other expression systems.

2. Inactivation by Serum Complement

Baculoviruses are rapidly inactivated by the complement system in serum.

This necessitates several modifications to reduce the negative impact of complement on baculovirus-mediated transduction.

The complement system is a common barrier for other gene delivery systems as well, including liposomes, murine retrovirus, and synthetic DNA complexes.

3. Fragility of Baculoviruses

The envelope structure, which includes the GP64 protein (GP64 is responsible for the attachment and entry of the virus inside the host cell via interaction with a not-very-well-characterized cellular receptor.) critical for viral and cellular membrane fusion, is essential for infectivity.

Baculoviruses are vulnerable to mechanical shear forces, leading to relatively low stability.

Purification via ultracentrifugation often results in significant loss of infectivity, likely due to damage to the viral envelopes.

Modest thermal stability further limits their usability, particularly in in vivo applications.

4. Reduced Stability in In Vivo Applications

The combination of modest thermal stability and susceptibility to serum complement reduces the viability of baculoviruses for in vivo gene delivery applications.

5. Limited Host Range

Baculoviruses primarily infect insect larvae and cells. However it does not naturally replicate in mammalian cells.

While they can transduce mammalian cells, this does not result in viral replication, limiting their broader application.

6. Suboptimal Glycosylation

Proteins expressed in insect cells often have glycosylation patterns that differ significantly from those of mammalian cells as insects cells have simple N-glycans with terminal mannose residues, while mammals have a wide variety of enzymes that result in more complex N-glycans with terminal sialic acid residues.

This can lead to improperly folded proteins or reduced bioactivity, especially for therapeutic proteins requiring human-like glycosylation.

References:

Pidre, M. L., Arrías, P. N., Amorós Morales, L. C., & Romanowski, V. (2022, December 28). The magic staff: A comprehensive overview of baculovirus-based technologies applied to human and Animal Health. Viruses.

https://pmc.ncbi.nlm.nih.gov/articles/PMC9863858/

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*Correspondence: victorbiol.unlp.edu.ar †These authors contributed equally to this work.

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The Magic Staff: A Comprehensive Overview of Baculovirus-Based Technologies Applied to Human and Animal Health

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Baculoviruses are enveloped, insect-specific viruses with large double-stranded DNA genomes. Among all the baculovirus species, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most studied. Due to its characteristics regarding…

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Challenges:

1. Expression levels vary greatly, with transmembrane proteins being the most difficult to produce. Proteins may misfold or be degraded, and differences in glycosylation between insect and mammalian cells can affect protein properties

Strategies:

(a) removing transmembrane region (TMR) of simple transmembrane proteins to create secreted forms

(b) Using different signal peptides to improve routing of proteins to cell surface, including signal peptides from honey bee melittin or baculovirus GP64

(c) Optimize codon usage & avoid long GC-rich sequences upstream of the ORF

2. Baculovirus genome instability can cause problems. Defective interfering particles with deletions including foreign gene, can form during continuous production, decreasing protein expression

Strategies:

(a) Infecting at low multiplicities of infection can reduce DI particle formation

(b) Inserting an extra copy of homologous region 1 (hr1) can stabilize foreign gene. An extra copy of hr1, combined with a deletion of the non-hr ori, resulted in stabilization of foreign gene expression

3. Viruses eventually kills host cells, making protein production non-continuous & require frequent re-infection of new cultures

Strategies:

(a) Using early or late viral promoters to express proteins earlier in the infection cycle, when cells & secretory pathway are likely still in good conditions

4. It requires multiple rounds of plaque purification, and some methods leave antibiotic marker genes, making this a time-consuming purification

Strategies:

(a) Using linearized baculovirus genomes at desired insertion site to increase he percentage of recombinants after recombination with the transfer plasmid. Employing bacmid technology allows for manipulation of the viral genome in E. coli, which uses site-directed transposition to introduce foreign genes and reduces the need for plaque purification

(b) Using semi-automated methods for generating recombinant viruses such as the flashBac system allows for high-throughput expression of foreign genes

References:

Author links open overlay panelMonique M. van Oers, AbstractIn this review background information on the baculovirus-insect cell expression system and its applications for producing protein subunits and virus-like particles for vaccine and other purposes is provided. This review will illustrate the princip, Ahrens, C. H., Ayres, M. D., Betenbaugh, M. J., Blanchard, P., Bouma, A., Burda, P., Carpentier, D. C., Chang, M., Cox, M. M. J., Fath-Goodin, A., Galibert, L., Guarino, L. A., Harper, D. M., Harrison, R. L., Hawtin, R. E., Hilton, S., Hitchman, R. B., … Condreay, J. P. (2011, July 22). Opportunities and challenges for the Baculovirus Expression System. Journal of Invertebrate Pathology. https://www.sciencedirect.com/science/article/pii/S0022201111000747?casa_token=0UPa7I3DkOEAAAAA%3AhObTiiN2kvBvIIMkWOLkxawshghroegytjRpqsp160xxOrlbozhBX0xDWNqm4LnaUfgnCRlXbM0#s0010

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Example 1: Cervarix vaccine [HPV16/18 L1 protein]

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Example 1: Cervarix vaccine [HPV16/18 L1 protein]

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Introduction:

Cervarix is a vaccine that was engineered with virus-like particles (VLPs) containing the L1 protein of HPV strains 16 and 18 [1]

The baculovirus expression system (BEVS) was used to synthesize the L1 proteins and transfected into insect cells

Cloning and expression:

With reference to the image attached, the gene of interest (GOI) is cloned into a plasmid [2]

In this case, the GOI would be the L1 gene which encodes for the L1 capsid proteins of HPV strains 16 and 18

A common transfer plasmid is the Autographa californica multiple-capsid nuclear polyhedrosis virus (AcMNPV [2]

The GOI will be inserted between the DNA of the AcMNPV and in order to ensure the virus becomes non-infectious, the polyhedrin promoter and the ORF1629 gene is removed [2].

Homologous recombination takes place to generate recombinant baculovirus (rBV)

The attached image shows how scale up processes take place to generate a pure plaque of rBV before transfecting them into insect cells to establish a working cell bank [2]

They can then be scaled up in a bioreactor/cell culture flasks

Purification:

After protein expression in culture flasks/bioreactors, the recombinant proteins can be purified by using ultra-centrifugation or column based chromatography [2]

In ultra centrifugation, high speeds are utilized to purify proteins

Due to the high G force, the proteins will separate with respect to their physical properties and molecular weight [3]

Hence, any impurities or contaminations can be easily separated from the POI

Additionally, protein tags such as His tags can be utilized to purify the proteins as well.

Downstream applications:

Production of these L1 proteins can be incorporated into vaccines for cervical cancer prevention

These recombinant L1 proteins will act as antigens against HPV 16 and 18, allowing the body to produce antibodies specific to these proteins

This prevents HPV infections in women and in turn, can prevent the formation of cervical cancer

References:

1. Hong, M., Li, T., Xue, W., Zhang, S., Cui, L., Wang, H., Zhang, Y., Zhou, L., Gu, Y., Xia, N., & Li, S. (2022). Genetic engineering of baculovirus-insect cell system to improve protein production. Frontiers in Bioengineering and Biotechnology, 10. https://doi.org/10.3389/fbioe.2022.994743

2. Felberbaum, R. S. (2015). The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors. Biotechnology Journal, 10(5), 702–714. https://doi.org/10.1002/biot.201400438

3. BiologyInsights Team. (2024, August 1). Ultracentrifugation: Principles, Types, and Applications Explained - BiologyInsights. BiologyInsights. https://biologyinsights.com/ultracentrifugation-principles-types-and-applications-explained/

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Example 2: HIV-1 Broadly Neutralizing Antibody b12

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Example 2: HIV-1 Broadly Neutralizing Antibody b12

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Introduction:

HIV-1 Broadly Neutralizing Antibody b12 is a monoclonal antibody that has been used to prevent the viral entry of HIV-1 virus [1]

This protein is produced using the baculovirus expression system as well

Cloning and expression:

The light and heavy chain genes of b12 need to be isolated and amplified by PCR [3]

The light chain gene for b12 is IGKV1-39 (variable region) and IGKJ1 (joining region) [2]

The heavy chain gene for b12 is IGHV1-3 [2]

Additionally, the signal peptide sequences for the Autographa californica 114 multiple nucleopolyhedrovirus (AcMNPV) was obtained and added to the 5' terminal of the heavy and light chain genes [3]

The pTrEx 1.1 vector was used to incorporate the final sequence in between the restriction sites of Ncol and Xhol in the MCS

In order to generate recombinant baculoviruses, the recombinant vector has to co-transfected together with the DNA of the bacmid in insect cells to allow homologous recombination to take place

The recombinant baculoviruses produced can then be used to infect insect cells to allow the expression of light and heavy chain proteins by placing them in shaker flasks for 3 days [3]

Purification:

To purify the produced proteins, bead-based chromatography and also sucrose gradient centrifugation [3]

Downstream applications:

Together with the VRC01 protein, the b12 antibody can be conjugated in a vaccine to act against HIV-1 infections

Since b12 binds to the CD4 binding site on HIV-1 glycoprotein envelopes, it acts as a barrier to prevent the virus from infecting hosts to propagate

References:

1. Wikipedia contributors. (2024, August 22). Broadly neutralizing HIV-1 antibodies. Wikipedia. https://en.wikipedia.org/wiki/Broadly_neutralizing_HIV-1_antibodies

2. GENETICS OF IMMUNOGLOBULINS. (n.d.). https://www.microbiologybook.org/mobile/m.immuno-6.htm

3. Liu, B., Wang, R., Wu, F., Xu, X., & Chen, H. (2014). Rapid production of HIV-1 neutralizing antibodies in baculovirus infected insect cells. Protein Expression and Purification, 99, 87–93. https://doi.org/10.1016/j.pep.2014.04.004