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Protein Expression and Purification Oct 2021Generating recombinant proteins in insect cells has been made possible via the use of the Baculovirus Expression Vector System (BEVS). Despite the success of many... (Review)
Review
Generating recombinant proteins in insect cells has been made possible via the use of the Baculovirus Expression Vector System (BEVS). Despite the success of many proteins via this platform, some targets remain a challenge due to issues such as cytopathic effects, the unpredictable nature of co-infection and co-expressions, and baculovirus genome instability. Many promoters have been assayed for the purpose of expressing diverse proteins in insect cells, and yet there remains a lack of implementation of those results when reviewing the landscape of commercially available baculovirus vectors. In advancing the platform to produce a greater variety of proteins and complexes, the development of such constructs cannot be avoided. A better understanding of viral gene regulation and promoter options including viral, synthetic, and insect-derived promoters will be beneficial to researchers looking to utilize BEVS by recruiting these intricate mechanisms of gene regulation for heterologous gene expression. Here we summarize some of the developments that could be utilized to improve the expression of recombinant proteins and multi-protein complexes in insect cells.
Topics: Animals; Baculoviridae; Cells, Cultured; Genetic Vectors; Insecta; Promoter Regions, Genetic; Recombinant Proteins; Sf9 Cells
PubMed: 34087362
DOI: 10.1016/j.pep.2021.105924 -
Protein Science : a Publication of the... Jan 2021The successful production of recombinant protein for biochemical, biophysical, and structural biological studies critically depends on the correct expression organism....
The successful production of recombinant protein for biochemical, biophysical, and structural biological studies critically depends on the correct expression organism. Currently, the most commonly used expression organisms for structural studies are Escherichia coli (~70% of all PDB structures) and the baculovirus/ insect cell expression system (~5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time-consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N- and C-terminal affinity, solubilization and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost-intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes.
Topics: Baculoviridae; Cloning, Molecular; Escherichia coli; Gene Expression; Genetic Vectors; Recombinant Proteins
PubMed: 32955754
DOI: 10.1002/pro.3957 -
Viruses Nov 2021The fall armyworm (FAW), , is a native pest species in the Western hemisphere. Since it was first reported in Africa in 2016, FAW has spread throughout the African... (Review)
Review
The fall armyworm (FAW), , is a native pest species in the Western hemisphere. Since it was first reported in Africa in 2016, FAW has spread throughout the African continent and is now also present in several countries in Asia as well as Australia. The invasion of FAW in these areas has led to a high yield reduction in crops, leading to huge economic losses. FAW management options in the newly invaded areas are limited and mainly rely on the use of synthetic pesticides. Since there is a risk of resistance development against pesticides in addition to the negative environmental and human health impacts, other effective, sustainable, and cost-efficient control alternatives are desired. Insect pathogenic viruses fulfil these criteria as they are usually effective and highly host-specific with no significant harmful effect on beneficial insects and non-target organisms. In this review, we discuss all viruses known from FAW and their potential to be used for biological control. We specifically focus on baculoviruses and describe the recent advancements in the use of baculoviruses for biological control in the native geographic origin of FAW, and their potential use in the newly invaded areas. Finally, we identify current knowledge gaps and suggest new avenues for productive research on the use of viruses as a biopesticide against FAW.
Topics: Animals; Baculoviridae; Biological Control Agents; Crops, Agricultural; Host Specificity; Insect Viruses; Pest Control, Biological; Spodoptera
PubMed: 34835026
DOI: 10.3390/v13112220 -
Viruses Dec 2022Baculoviruses are enveloped, insect-specific viruses with large double-stranded DNA genomes. Among all the baculovirus species, Autographa californica multiple... (Review)
Review
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 biosafety, narrow host range and the availability of different platforms for modifying its genome, AcMNPV has become a powerful biotechnological tool. In this review, we will address the most widespread technological applications of baculoviruses. We will begin by summarizing their natural cycle both in larvae and in cell culture and how it can be exploited. Secondly, we will explore the different baculovirus-based protein expression systems (BEVS) and their multiple applications in the pharmaceutical and biotechnological industry. We will focus particularly on the production of vaccines, many of which are either currently commercialized or in advanced stages of development (e.g., Novavax, COVID-19 vaccine). In addition, recombinant baculoviruses can be used as efficient gene transduction and protein expression vectors in vertebrate cells (e.g., BacMam). Finally, we will extensively describe various gene therapy strategies based on baculoviruses applied to the treatment of different diseases. The main objective of this work is to provide an extensive up-to-date summary of the different biotechnological applications of baculoviruses, emphasizing the genetic modification strategies used in each field.
Topics: Animals; Humans; Baculoviridae; COVID-19 Vaccines; COVID-19; Nucleopolyhedroviruses; Spodoptera
PubMed: 36680120
DOI: 10.3390/v15010080 -
Annual Review of Entomology Jan 2023Silkworm () is not only an economic insect but also a model organism for life science research. nucleopolyhedrovirus (BmNPV) disease is a major infectious disease in... (Review)
Review
Silkworm () is not only an economic insect but also a model organism for life science research. nucleopolyhedrovirus (BmNPV) disease is a major infectious disease in the world's sericulture industry. The cocoon loss caused by this disease accounts for more than 60% of the total loss caused by all silkworm diseases. To date, there has been no effective solution for preventing and treating this disease. The most effective measure is to breed disease-resistant varieties. The quickest way to breed disease-resistant varieties is to apply genetic modification. However, this requires that we obtain disease resistance genes and know the mechanism of disease resistance. Since the discovery of disease-resistant resources in 1989, scholars in the sericulture industry around the world have been inspired to search for resistance genes. In the past two decades, with the help of multi-omics technologies, screening of resistance genes, gene localization, protein modification, virus-host interactions, etc., researchers have found some candidate genes that have been proposed to function at the cellular or individual level. Several disease-resistant varieties have been obtained and used in production through hybrid breeding, RNA interference, and genetic modification. This article summarizes and reviews the discovery of and research advances related to silkworm resistance to BmNPV. It is anticipated that the review will inspire scientific researchers to continue searching for disease resistance genes, clarify the molecular mechanism of silkworm disease resistance, and promote disease-resistant silkworm breeding.
Topics: Animals; Baculoviridae; Bombyx; Disease Resistance; Nucleopolyhedroviruses
PubMed: 36689303
DOI: 10.1146/annurev-ento-120220-112317 -
BMC Biology Oct 2017The MultiBac baculovirus/insect cell expression vector system was conceived as a user-friendly, modular tool-kit for producing multiprotein complexes for structural... (Review)
Review
The MultiBac baculovirus/insect cell expression vector system was conceived as a user-friendly, modular tool-kit for producing multiprotein complexes for structural biology applications. MultiBac has allowed the structure and function of many molecular machines to be elucidated, including previously inaccessible high-value drug targets. More recently, MultiBac developments have shifted to customized baculoviral genomes that are tailored for a range of applications, including synthesizing artificial proteins by genetic code expansion. We review some of these developments, including the ongoing rewiring of the MultiBac system for mammalian applications, notably CRISPR/Cas9-mediated gene editing.
Topics: Animals; Baculoviridae; CRISPR-Cas Systems; Gene Editing; Genome, Viral; Mammals; Molecular Biology; Multiprotein Complexes
PubMed: 29084535
DOI: 10.1186/s12915-017-0447-6 -
Viruses Apr 2023Accurate and rapid quantification of (infectious) virus titers is of paramount importance in the manufacture of viral vectors and vaccines. Reliable quantification data...
Accurate and rapid quantification of (infectious) virus titers is of paramount importance in the manufacture of viral vectors and vaccines. Reliable quantification data allow efficient process development at a laboratory scale and thorough process monitoring in later production. However, current gold standard applications, such as endpoint dilution assays, are cumbersome and do not provide true process analytical monitoring. Accordingly, flow cytometry and quantitative polymerase chain reaction have attracted increasing interest in recent years, offering various advantages for rapid quantification. Here, we compared different approaches for the assessment of infectious viruses, using a model baculovirus. Firstly, infectivity was estimated by the quantification of viral nucleic acids in infected cells, and secondly, different flow cytometric approaches were investigated regarding analysis times and calibration ranges. The flow cytometry technique included a quantification based on post-infection fluorophore expression and labeling of a viral surface protein using fluorescent antibodies. Additionally, the possibility of viral (m)RNA labeling in infected cells was investigated as a proof of concept. The results confirmed that infectivity assessment based on qPCR is not trivial and requires sophisticated method optimization, whereas staining of viral surface proteins is a fast and feasible approach for enveloped viruses. Finally, labeling of viral (m)RNA in infected cells appears to be a promising opportunity but will require further research.
Topics: Baculoviridae; Genetic Vectors; RNA
PubMed: 37112978
DOI: 10.3390/v15040998 -
Virus Research Dec 2022Transfer RNAs (tRNAs) genes are both coded for and arranged along some viral genomes representing the entire virosphere and seem to play different biological functions...
Transfer RNAs (tRNAs) genes are both coded for and arranged along some viral genomes representing the entire virosphere and seem to play different biological functions during infection, other than transferring the correct amino acid to a growing peptide chain. Baculovirus genome description and annotation has focused mostly on protein-coding genes, microRNA, and homologous regions. Here we carried out a large-scale in silico search for putative tRNA genes in baculovirus genomes. Ninety-six of 257 baculovirus genomes analyzed was found to contain at least one putative tRNA gene. We found great diversity in primary and secondary structure, in location within the genome, in intron presence and size, and in anti-codon identity. In some cases, genes of tRNA-containing genomes were found to have a bias for the codons specified by the tRNAs present in such genomes. Moreover, analysis revealed that most of the putative tRNA genes possessed conserved motifs for tRNA type 2 promoters, including the A-box and B-box motifs with few mismatches from the eukaryotic canonical motifs. From publicly available small RNA deep sequencing datasets of baculovirus-infected insect cells, we found evidence that a putative Autographa californica multiple nucleopolyhedrovirus Gln-tRNA gene was transcribed and modified with the addition of the non-templated 3'-CCA tail found at the end of all tRNAs. Further research is needed to determine the expression and functionality of these viral tRNAs.
Topics: Baculoviridae; RNA, Transfer; Eukaryota; Base Sequence; Codon
PubMed: 36181979
DOI: 10.1016/j.virusres.2022.198949 -
Journal of Pharmacological Sciences Mar 2023The outbreak of the SARS-2-CoV infection has become a global outbreak and continues to cause many deaths. In addition, the risk of pandemics continues to increase due to... (Review)
Review
The outbreak of the SARS-2-CoV infection has become a global outbreak and continues to cause many deaths. In addition, the risk of pandemics continues to increase due to environmental changes and the globalization of human exchange and logistics. On the other hand, our preparedness for emerging infectious diseases caused by such unknown viruses is inadequate, and dealing with viral infections is one of the most important issues that need to be addressed immediately. Vaccine based disease control is considered an ideal countermeasure for infectious diseases, as it is expected to provide maximum efficacy at minimum cost. Although new nucleic acid-based vaccines are leading the way in the prevention of COVID-19, the mainstream of vaccines is still inactivated or live attenuated vaccines that use the pathogen virus itself. Subunit vaccines, in which specific virus-derived proteins are produced as recombinant proteins and used as vaccine antigens, have been developed, but production and development of many antigens that are difficult to mass-produce as recombinant proteins, such as the spike protein antigen of COVID-19 has not progressed. This paper describes the development of recombinant protein vaccines using the silkworm, which has an advantage in the production of such difficult-to-express vaccine antigens, especially virus-like particles.
Topics: Animals; Humans; Bombyx; Viral Vaccines; Antiviral Agents; COVID-19; Recombinant Proteins; Baculoviridae
PubMed: 36828618
DOI: 10.1016/j.jphs.2023.01.002 -
Journal of Biomolecular NMR Jul 2018Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article... (Review)
Review
Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production of recombinant isotope-labeled proteins. We discuss recent advances in inexpensive isotope-labeling methods using labeled algal or yeast extracts as the amino acid source and give examples of advanced NMR applications for proteins, which have become accessible by this eukaryotic expression host.
Topics: Animals; Baculoviridae; Humans; Insecta; Isotope Labeling; Nuclear Magnetic Resonance, Biomolecular; Proteins; Recombinant Proteins
PubMed: 29687312
DOI: 10.1007/s10858-018-0172-7