Proteins In Insect Cells Research Articles

Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and ultimately affect viral replication.

Alternative Strategies for Expressing Multicomponent Protein Complexes in Insect Cells.

Expression of recombinant proteins in insect cells infected with baculoviruses is commonplace. This system provides an easy way to generate a significant amount of properly folded, functional protein with proper posttranslational modifications and can be used to effectively produce multi-protein complexes. This chapter describes an alternative method of expressing high order protein complexes in insect cell culture. Specific examples involving the expression of 5- and 8-protein complexes are discussed.

Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System.

P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed proteins can be isolated. Insect cells are a perfect system to study P-type ATPases as they have little or no endogenous Na,K-ATPase activity and other ATPase activities can be inhibited easily. Here we describe in detail the expression and isolation of Na,K-ATPase and H,K-ATPase isoforms with the baculovirus expression system.

Fundamentals of Baculovirus Expression and Applications.

In 1982 E. coli produced human insulin, the world's first recombinant DNA drug, was approved by the FDA. Since this historical event, remarkable progress has been made in developing bacterial, yeast, mammalian and insect cell protein expression systems that are used to produce recombinant proteins for both research and clinical applications. Of the available approaches, the insect cell based baculovirus expression vector system (BEVS) has proven to be a particularly adaptable system for producing a diverse collection of proteins. Along with E. coli, the system has been valuable for the production of proteins for structural studies, including adequate quantities of difficult to produce G protein-coupled receptors. BEVS has also been used for production of the human papilloma virus vaccine, Cervarix, the first FDA approved insect cell produced product and FluBlok, a vaccine based on the influenza virus hemagglutinin protein. Baculoviruses, modified to contain mammalian promoters (BacMam viruses), have proven to be efficient gene delivery vectors for mammalian cells and provide an alternative transient mammalian cell based protein expression approach to that of plasmid DNA based transfection methodologies. Here we provide an update on recent advances in baculovirus vector development with a focus on the numerous applications of these viruses in basic research and biotechnology.

Codon Usage in Signal Sequences Affects Protein Expression and Secretion Using Baculovirus/Insect Cell Expression System

By introducing synonymous mutations into the coding sequences of GP64sp and FibHsp signal peptides, the influences of mRNA secondary structure and codon usage of signal sequences on protein expression and secretion were investigated using baculovirus/insect cell expression system. The results showed that mRNA structural stability of the signal sequences was not correlated with the protein production and secretion levels, and FibHsp was more tolerable to codon changes than GP64sp. Codon bias analyses revealed that codons for GP64sp were well de-optimized and contained more non-optimal codons than FibHsp. Synonymous mutations in GP64sp sufficiently increased its average codon usage frequency and resulted in dramatic reduction of the activity and secretion of luciferase. Protein degradation inhibition assay with MG-132 showed that higher codon usage frequency in the signal sequence increased the production as well as the degradation of luciferase protein, indicating that the synonymous codon substitutions in the signal sequence caused misfolding of luciferase instead of slowing down the protein production. Meanwhile, we found that introduction of more non-optimal codons into FibHsp could increase the production and secretion levels of luciferase, which suggested a new strategy to improve the production of secretory proteins in insect cells.

Abstract A148: Developing a baculovirus expression system to evaluate the role of B2-glycoprotein I in anti-tumor cell growth

Abstract β2-glycoprotein I (β2-GPI) is a human plasma protein which has been reported to elicit an anti-cell migration effect on human aortic endothelial cells in our previous study. Migration of endothelial cells has been found to be involved in tumorigenesis, however, whether β2-GPI regulates tumor cell growth is unknown. In this study, we investigated the biological effect of β2-GPI on anti-cell migration and proliferation in tumor cells using a baculovirus expression system. Based on Bac-to-Bac Baculovirus Expression system, the cDNA encoding B2-GPI gene has been constructed and cloned into the pFastBac vector with additionally designed sequences, including a Honeybee Melittin Secretion Signal, a dual Flag/StrepII affinity tag and a Tabacco Etch Virus cleavage site at N terminal end and a C-terminal IgG1 Fc fusion protein sequences. This recombinant bacmid DNA containing a complete B2-GPI gene was transfected into insect Sf9 cells to generate recombinant baculovirus. Expression of these chimeric proteins in insect cells resulted in efficient assembly and release of hybrid recombinant B2-GPI protein into cultured media. The fusion proteins containing IgG1-Fc sequences can be purified by the protein A affinity column. Finally, we collected the recombinant B2-GPI in high purity and expression yield. Direct administration of recombinant B2-GPI protein resulted in an effective inhibition of the melanoma cell migration and tumor growth. Development of this baculoviral-based system provides an option for investigation in the anti-tumor cell growth function of B2-GPI in vitro and in vivo. Our study provides a baculoviral-based approach which opens a new perspective for developing B2-GPI as an agent in anti-cancer therapy. Citation Format: Yu-Shan Lin, Shr-Jeng Leu, An-Na Chiang. Developing a baculovirus expression system to evaluate the role of B2-glycoprotein I in anti-tumor cell growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A148.

Crimean-Congo Hemorrhagic Fever Virus Gn Bioinformatic Analysis and Construction of a Recombinant Bacmid in Order to Express Gn by Baculovirus Expression System.

BackgroundCrimean-Congo hemorrhagic fever virus (CCHFV) is a member of the nairovirus, a genus in the Bunyaviridae family, which causes a life threatening disease in human. Currently, there is no vaccine against CCHFV and detailed structural analysis of CCHFV proteins remains undefined. The CCHFV M RNA segment encodes two viral surface glycoproteins known as Gn and Gc. Viral glycoproteins can be considered as key targets for vaccine development.ObjectivesThe current study aimed to investigate structural bioinformatics of CCHFV Gn protein and design a construct to make a recombinant bacmid to express by baculovirus system.Materials and MethodsTo express the Gn protein in insect cells that can be used as antigen in animal model vaccine studies. Bioinformatic analysis of CCHFV Gn protein was performed and designed a construct and cloned into pFastBacHTb vector and a recombinant Gn-bacmid was generated by Bac to Bac system.ResultsPrimary, secondary, and 3D structure of CCHFV Gn were obtained and PCR reaction with M13 forward and reverse primers confirmed the generation of recombinant bacmid DNA harboring Gn coding region under polyhedron promoter.ConclusionsCharacterization of the detailed structure of CCHFV Gn by bioinformatics software provides the basis for development of new experiments and construction of a recombinant bacmid harboring CCHFV Gn, which is valuable for designing a recombinant vaccine against deadly pathogens like CCHFV.

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig.

Expression and purification of HER2 extracellular domain proteins in Schneider2 insect cells

Overexpression of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) results in ligand independent activation of kinase signaling and is found in about 30% of human breast cancers, and is correlated with a more aggressive tumor phenotype. The HER2 extracellular domain (ECD) consists of four domains − I, II, III and IV. Although the role of each domain in the dimerization and activation of the receptor has been extensively studied, the role of domain IV (DIV) is not clearly understood yet. In our previous studies, we reported peptidomimetic molecules inhibit HER2:HER3 heterodimerization. In order to study the binding interactions of peptidomimetics with HER2 DIV in detail, properly folded recombinant HER2 protein in pure form is important. We have expressed and purified HER2 ECD and DIV proteins in the Drosophila melanogaster Schneider2 (S2) cell line. Using the commercial Drosophila expression system (DES), we transfected S2 cells with plasmids designed to direct the expression of secreted recombinant HER2 ECD and DIV proteins. The secreted proteins were purified from the conditioned medium by filtration, ultrafiltration, dialysis and nickel affinity chromatography techniques. The purified HER2 proteins were then analyzed using Western blot, mass spectrometry and circular dichroism (CD) spectroscopy.

Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins-especially those entering the secretory pathway- provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

Expression of porcine circovirus type 2 capsid protein fused with partial polyhedrin using baculovirus

Porcine circovirus type 2 (PCV2) is an important infectious swine virus causing postweaning multisystemic wasting syndrome (PMWS). PCV2 capsid protein, encoded by ORF2 has typespecific epitopes, is very immunogenic, and is associated with the induction of neutralizing antibodies. For the efficient production of capsid protein, recombinant Autographa californica nucleopolyhedroviruses were generated to express ORF2 fused with two forms of a partial polyhedrin. Recombinant capsid protein was produced successfully with the partial polyhedrin fusion form and the yield was high, as was shown by SDS-PAGE. Production of recombinant capsid proteins in insect cells was confirmed by Western blot analysis using anti- His monoclonal antibody, anti-ORF2 monoclonal antibody, and anti-PCV2 porcine serum. Fusion expression with amino acids 19 to 110 of the polyhedrin increased the production of recombinant capsid protein, but fusion with amino acids 32 to 85 did not. Additionally, PCV2 capsid protein is a glycoprotein; however, the glycosylation of recombinant protein was not observed. The results of an Enzyme-linked immunosorbent assay (ELISA) showed that recombinant capsid proteins could be utilized as antigens for fast, large-scale diagnosis of PCV2-infected pigs. Our results suggest that the fusion expression of partial polyhedrin is able to increase the production of recombinant PCV2 capsid protein in insect cells.

An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts.

Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein (15)N and (13)C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor.

The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

Small interfering (Si) RNA mediated baculovirus replication reduction without affecting target gene expression

The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant protein with posttranslational modification. Recombinant baculoviruses (such as Autographa californica multiple nuclear polyhedrosis virus) are especially useful in producing recombinant proteins and virus-like particles (VLPs) as biodrugs or candidate vaccines for the prevention of serious infectious diseases. However, during the bioprocessing of recombinant proteins in insect cells, baculovirus replication and viral budding are coincident. In some cases, residual baculovirus contaminants remain in the recombinant protein products, even though various purification processes are applied such as ion-exchange chromatography, ultracentrifugation, or gel filtration. To reduce unexpected contamination caused by replication and budding-out of the baculovirus, we designed short interfering (si) RNAs targeting glycoprotein 64 (GP64) or single-stranded DNA-binding protein (DBP) to inhibit baculovirus replication during overexpression of recombinant foreign genes. GP64 is known to be critical both for the entry of virions into cells and for the assembly of the budded virion at the cell surface. DBP is also essential for virus assembly by regulation of the capsid protein P39 and the polyhedrin protein. This study showed that GP64 expression was suppressed by GP64 siRNAs in Western blot experiments, while the expression of recombinant proteins was unaffected. In addition, transfection of GP64 siRNAs and DBP siRNAs reduced the level of baculovirus replication, compared with the treatment with scrambled siRNAs. However, DBP siRNA also suppressed the expression of recombinant proteins. In conclusion, our GP64 siRNAs showed that an interfering RNA system, such as siRNAs and short hairpin (sh) RNAs, can be applicable to reduce baculovirus contaminants during the bioprocessing of recombinant proteins in insect cells. Further investigation should be carried out to establish transformed insect cell lines with stable expression of corresponding interfering RNAs.

Isotope labeling of proteins in insect cells.

Protein targets of contemporary research are often membrane proteins, multiprotein complexes, secreted proteins, or other proteins of human origin. These are difficult to express in the standard expression host used for most nuclear magnetic resonance (NMR) studies, Escherichia coli. Insect cells represent an attractive alternative, since they have become a well-established expression system and simple solutions have been developed for generation of viruses to efficiently introduce the target protein DNA into cells. Insect cells enable production of a larger fraction of the human proteome in a properly folded way than bacteria, as insect cells have a very similar set of cytosolic chaperones and a closely related secretory pathway. Here, the limited and defined glycosylation pattern that insect cells produce is an advantage for structural biology studies. For these reasons, insect cells have been established as the most widely used eukaryotic expression host for crystallographic studies. In the past decade, significant advancements have enabled amino acid type-specific as well as uniform isotope labeling of proteins in insect cells, turning them into an attractive expression host for NMR studies.

Characterization of the Glycan Structures of GlycoproteinGA733‐Fc Expressed in a Baculovirus‐Insect Cell System

Baculovirus‐insect cell systems have been used to express functional recombinant biopharmaceutical proteins. TwopFastBacDual vectors carrying the gene encoding antigenGA733, a cell‐surface glycoprotein, fused to theIgGFc (GA733‐Fc) orKDEL(endoplasmic reticulum [ER] retention sequence) (GA733‐FcK) genes were constructed to generate baculoviruses expressing the corresponding recombinant proteins in insect cells. The expression of the recombinantGA733‐Fc andGA733‐FcKproteins and their glycan structure profiles were assessed under various conditions by analyzing the cell line (Sf9 and High Five), the postinfection (PI) time (48, 72, and 96 h), and the harvested sample (cell culture media [CM] or cell lysate [CL]). Immunoblotting showed nonidentical expression of bothGA733‐Fc andGA733‐FcKunder various conditions. Glycosylation analysis revealed that varying conditions affected the glycan structure profiles. These results suggest that thePItime, subcellular localization, and cell type affect recombinant protein expression as well as glycosylation in the baculovirus‐insect cell system.

The assembly of virus-like particles of porcine transmissible gastroenteritisvirus in vitro by baculovirus expression system

Virus-like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for transmissible gastroenteritis virus (TGEV) assembly and release. In this study, the M gene, sM gene, and N gene of TGEV were inserted into donor plasmid pFastBac Dual. Those recombinant donor plasmids were transformed into E.coli DH10Bac respectively. Recombinant baculoviruses were obtained by transfection of the Sf9 cells with Bacmid-M, Bacmid-sM, and Bacmid-N. The results of indirect immunofluorescence assay showed that recombinant M protein, sM protein, and N protein were expressed successfully. In vitro experiments of VLP assembly of TGEV were performed by infection of insect cells with various combinations of recombinant baculoviruses identified. It is shown that expression of M protein alone, coexpression of M plus sM protein, and coexpression of M plus N protein in Sf9 insect cells infected by recombinant baculovirus could result in the occurrence of a virion morphologically similar to TGEV. However, the sM and N protein could not generate a structure similar to the virion of TGEV when expressed alone. This study provides a theoretical foundation for further study on the role of TGEV structural proteins in the assembly process of virions.

Thirty years of baculovirus–insect cell protein expression: from dark horse to mainstream technology

In December 1983, a seminal paper appeared on the overexpression of human IFN-β in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produced massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes were dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years, major improvements were achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted BEVs are used to deliver and express heterologous genes in mammalian cells, and they may be used for gene therapy and cancer treatment in the future. The purpose of this review is to highlight the thirtieth 'anniversary' of this expression system by summarizing the fundamental research and major technological advances that allowed its development, whilst noting challenges for further improvements.

Peste des petits ruminants virus-like particles induce both complete virus-specific antibodies and virus neutralizing antibodies in mice

Peste des petits ruminants virus (PPRV), an etiological agent of peste des petits ruminants (PPR), is classified into the genus Morbillivirus in the family Paramyxoviridae. In a previous study, a recombinant baculovirus has been constructed to co-express the PPRV matrix (M), haemagglutinin (H) and nucleocapsid (N) proteins in insect cells, causing budding of PPR virus-like particles (VLPs) from insect cell membranes by viewing of ultrathin section with a transmission electron microscope. In this follow-up study, these PPR VLPs were purified by sucrose density gradient centrifugation for immunizing mice twice. Three weeks post-primary immunization and 2 weeks post-secondary immunization, all serum samples were obtained and subsequently subjected to indirect ELISA detection on complete virus-specific antibodies. In addition, all serum samples, which were collected 2 weeks post-secondary immunization, were used for virus neutralization test on PPRV neutralizing antibodies. The results showed that the purified PPR VLPs induced both types of antibodies mentioned above in mice, indicating a given potential of VLP-based vaccine candidate against PPR.

GFP-based expression screening of membrane proteins in insect cells using the baculovirus system.

A key step in the production of recombinant membrane proteins for structural studies is the optimization of protein yield and quality. One commonly used approach is to fuse the protein to green fluorescent protein (GFP), enabling expression to be tracked without the need to purify the protein. Combining fusion to green fluorescent protein with the baculovirus expression system provides a useful platform for both screening and production of eukaryotic membrane proteins. In this chapter we describe our protocol for the expression screening of membrane proteins in insect cells using fusion to GFP as a reporter. We use both SDS-PAGE with in-gel fluorescence imaging and fluorescence-detection size-exclusion chromatography (FSEC) to screen for expression.