Recombinant Proteins In Insect Cells Research Articles

Cell Culture Media Optimization for Increased Production of Recombinant Proteins in Insect Cells

The insect cell/baculovirus expression system (IC-BEVS) has been at the forefront of biotechnologicalresearch, and has served as a tool to produce several human therapeutics such asCervavix ® (preventative vaccine against the human papilloma Virus), Flublok ® (preventativevaccine against seasonal influenza virus), Provenge ® (therapeutic treatment against prostatecancer) and Glybera ® (gene therapy treatment for lipoprotein lipase deficiency). IC-BEVS isan attractive alternative to mammalian cells for biomanufacturing as it offers advantages suchas easy adaptation to serum-free media, high levels of protein expression and post-translationalmodifications, and is appropriately scalable for manufacturing 2. Despite these advantages,the use of IC-BEVS to produce recombinant proteins can be costly and time-consuming.As the demand for new therapeutic increases, efficient, and robust methods to improvethe production and screening processes of recombinant proteins in this expression systemare necessary to respond to large-scale manufacturing needs. Research has shown that tailoredmedia supplementation and optimization is an efficient, and useful strategy to reachhigh density cultures and increase protein production in-vitro. Therefore, this project aimed toevaluate the effects of two macromolecules (glucose in high concentration, and glutathione)as potential cell culture additives or boosters to increase the production of therapeutic proteinsin the baculovirus/insect cell expression system. It was demonstrated that glutathioneaddition resulted in a more rapid, universal production of protein compared to minimal media,suggesting this to be a valuable addition to IC-BEVS media.

Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells

Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors.

Effects of autophagy inducers on recombinant antibody production in insect cells.

Insect cells have recently proven to be an excellent platform for the high-level production of functional recombinant proteins. Autophagy is an important mechanism that promotes cell survival by eliminating damaged organelles and protein aggregates, and it also may influence recombinant protein production. In the present study, we compared the effects that autophagy inducers rapamycin, everolimus, and lithium chloride exert on recombinant lepidopteran insect cells that secrete an engineered antibody molecule. Compared with nontreatment, treatment with either rapamycin or everolimus prolonged cell growth to allow high cell density, improved viability in the declining phase, and then increased the yield of secreted antibodies. These positive effects appeared to be induced via autophagy since autophagosomes were clearly detected, particularly in cells treated with rapamycin or everolimus. Unlike rapamycin, another autophagy inducer, FK506, was ineffective in insect cells. The addition of an appropriate autophagy inducer may be effective in increasing the productivity of recombinant proteins in insect cells.

A scalable suspension insect cell transfection method for production of baculoviruses with low amplification passages

Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer of Spodoptera frugiperda insect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180 bp to 2,395 bp, were obtained through employment of the Bac-to-Bac expression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6 × 106 cells, the recombinant baculoviruses titer obtained with modified method was about 2 × 107 pfu/ mL in a total volume of 12 mL, which is scalable to 24 liters of 1 × 108 pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages.

A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

Characterization of the honeybee venom proteins C1q-like protein and PVF1 and their allergenic potential

Honeybee (Apis mellifera) venom (HBV) represents an ideal model to study the role of particular venom components in allergic reactions in sensitized individuals as well as in the eusociality of Hymenoptera species. The aim of this study was to further characterize the HBV components C1q-like protein (C1q) and PDGF/VEGF-like factor 1 (PVF1). C1q and PVF1 were produced as recombinant proteins in insect cells. Their allergenic properties were examined by determining the level of specific IgE antibodies in the sera of HBV-allergic patients (n = 26) as well as by their capacity to activate patients' basophils (n = 11). Moreover, the transcript heterogeneity of PVF1 was analyzed. It could be demonstrated that at least three PVF1 variants are present in the venom gland, which all result from alternative splicing of one transcript. Additionally, recombinant C1q and PVF1 from Spodoptera frugiperda insect cells exhibited specific IgE reactivity with approximately 38.5% of sera of HBV-allergic patients. Interestingly, both proteins were unable to activate basophils of the patients, questioning their role in the context of clinically relevant sensitization. Recombinant C1q and PVF1 can build the basis for a deeper understanding of the molecular mechanisms of Hymenoptera venoms. Moreover, the conflicting results between IgE sensitization and lack of basophil activation, might in the future contribute to the identification of factors that determine the allergenic potential of proteins.

Overview of the Baculovirus Expression System

This unit provides information on the replication cycle of insect baculovirus to provide an understanding of how this virus has been adapted for use as an expression vector for recombinant proteins in insect cells. We provide an overview of the virus structure and its unique bi-phasic replication cycle, which has been exploited in developing the virus as an expression vector. We also review the development of the baculovirus expression vector system (BEVS), from the mid-1980s to the present day in which the BEVS is now an established tool for the production of a range of recombinant proteins and multi-protein complexes including virus-like particles. We describe advances made to the BEVS to allow the rapid and easy production of recombinant viruses and developments to improve protein yield. We finish by describing the application of recombinant BacMam as vectors for the delivery of genes into mammalian and human cells. © 2018 by John Wiley & Sons, Inc.

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.

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.

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.

Characterization of the Glycan Structures of Glycoprotein GA733‐Fc Expressed in a Baculovirus‐Insect Cell System

Baculovirus‐insect cell systems have been used to express functional recombinant biopharmaceutical proteins. Two pFastBac Dual vectors carrying the gene encoding antigen GA733, a cell‐surface glycoprotein, fused to the IgG Fc (GA733‐Fc) or KDEL (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 recombinant GA733‐Fc and GA733‐FcK proteins 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 both GA733‐Fc and GA733‐FcK under various conditions. Glycosylation analysis revealed that varying conditions affected the glycan structure profiles. These results suggest that the PI time, subcellular localization, and cell type affect recombinant protein expression as well as glycosylation in the baculovirus‐insect cell system.

Expression of recombinant proteins in insect cells by their direct infection with Escherichia coli transformed with baculovirus bacmids

The baculovirus-insect cell expression system (BES), one of the most popular systems for expression of eukaryotic proteins, was known to have drawbacks such as laborious manipulation of large-size baculovirus bacmids and the transfection procedure. These problems could be eliminated by direct infection of eukaryotic cells with nonpathogenic bacteria harbouring the respective gene - bactofection. However, it was unknown whether this system could be applied to insect cells. Therefore, in this study, the possibility of delivery of enhanced green fluorescent protein (EGFP) gene as a marker into the insect cell lines Sf9 and BmN-SWU1 using the above-mentioned approach with the Bac-to-Bac system was investigated. Using a simple co-incubation of Escherichia coli strains containing recombinant baculovirus bacmids with the EGFP gene and insect cells, it was possible to demonstrate the EGFP expression in these cells and to obtain high-titer recombinant baculoviral stocks. Furthermore, BmN-SWU1 cells proved more susceptible to the used E. coli strains than Sf9 cells. However, the co-expression of invasin and listeriolysin-O, known to enhance the E. coli-mediated gene delivery to mammalian cells, with EGFP, had no effect on insect cells. Summing up, this study proved that a heterologous gene can be efficiently delivered and expressed in insect cells by their simple incubation with non-pathogenic E. coli strains harboring recombinant baculovirus bacmids with the respective gene.

PRODUCTION OF RECOMBINANT PROTEINS IN INSECT CELLS

Among the wide range of methods and expression hosts available for the heterologous production of recombinant proteins, insect cells are ideal for the production of complex proteins requiring extensive post-translational modification. This review article provides an overview of the available insect-cell expression systems and their properties, focusing on the widely-used Baculovirus Expression Vector System (BEVS). We discuss the different strategies used to generate recombinant baculovirus vectors and show how advanced techniques for virus titer determination can accelerate the production of recombinant proteins. The stable transfection of insect cells is an alternative to BEVS which has recently been augmented with recombinase-mediated cassette exchange for site-specific gene integration. We consider the advantages and limitations of these techniques for the production of recombinant proteins in insect cells and compare them to other expression platforms.

α-Synuclein and β-synuclein enhance secretion protein production in baculovirus expression vector system

The baculovirus expression vector system (BEVS) is widely used as a tool for expressing of recombinant proteins in insect cells or larvae. However, the expression level of secretion pathway proteins is often lower than that of cytosolic and nucleus proteins. Thus, we attempted to improve production of secreted proteins by using a secretory alkaline phosphatase-EGFP fusion protein (SEFP)-based bi-cistronic baculovirus vector to identify chaperones that have potential on boosting secreted protein production. As co-expressed SEFP with a chaperone, calreticulin (CALR), it was found that the secreted SEFP enzyme activity can be boosted up to twofold. This result demonstrated the SEFP-based bi-cistronic approach can be used to identify the genes that can enhance secretion protein production in BEVS. Thus, the chaperone activity of α-synuclein (α-syn) and β-synuclein (β-syn) was evaluated in cells co-expressed with SEFP and compared that with CALR by analyzing localization, alkaline phosphatase enzyme activity, and mRNA expression levels of SEFP. Our results showed that SEFP enzyme activity from cells co-expressed with both synuclein proteins can be enhanced up to 2.3-fold and this increment was better than that caused by CALR. Moreover, this enhancement might arise from the transcription enhancement or higher RNA stability. By this novel approach, we provided evidences that α- and β-syn can enhance secretion proteins production in BEVS.

Baculovirus as a vaccine vector

Baculovirus is extensively utilized as an excellent tool for production of recombinant protein in insect cells. Baculovirus infects insects in nature and is non-pathogenic to humans. In addition to insect cells, baculovirus is capable of transducing a broad range of animal cells. Due to its biosafety, large cloning capacity, low cytotoxicity, and non-replication nature in the transduced cells as well as the ease of manipulation and production, baculovirus has been utilized as RNA interference mediators, gene delivery vectors, and vaccine vectors for a wide variety of applications. This article focuses on the utilization of baculoviruses as vaccine vectors to prepare antigen or subunit vaccines.

Efficient Production of Porcine Circovirus Type 2 Capsid Protein using Baculovirus

Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus associated with Postweaning multisystemic wasting syndrome (PMWS), which is considered to be an important infectious swine viral disease. PCV2 capsid protein encoded by ORF2 is a structural protein and expected as the high immunogenicity protein. In this study, we generated recombinant baculovirus containing ORF2 of PCV2 and analyzed the optimal conditions for the production of capsid protein in insect cell. Production and status of recombinant capsid protein in insect cell were confirmed by SDS-PAGE and Western blot analysis using His tag antibody and anti-PCV2 serum. The yield of recombinant capsid protein was high like as shown visible on SDS-PAGE. Optimal multiplicity of infection (MOI) and infection time of recombinant virus were determined as 5 MOI and 4 days, respectively. ORF2 is known to have N-linked glycosylation site, but we couldn't detect the glycosylation of recombinant protein in insect cells.

Expression of recombinant proteins in plants by using baculovirus vectors

Baculovirus has been widely used for the production of numerous recombinant proteins in insect cells. Baculovirus vectors have several advantages, including proper post-translational modification, biosafety, and multiple large gene expression ability. Most insect cell-produced proteins have been expressed by using the baculovirus expression vector system (BEVS) under the control of strong polyhedrin (Polh) or p10 promoters. There has been no report on the expression of recombinant proteins by baculovirus in plant cells. In this study, we used the baculovirus vector to express recombinant green fluorescent protein (GFP) in plants. To investigate the expression of GFP protein by baculovirus in plants, we cloned the gfp gene under the control of Polh promoter or Cauliflower Mosaic Virus (CaMV) 35S promoter to yield the Polh-GFP and 35S- GFP bacmids carrying the GFP expression cassettes, respectively. The presence of Polh-GFP and 35S-GFP expression cassettes in the bacmids was confirmed by polymerase chain reaction (PCR). Subsequently, both the GFP bacmids and GFP baculovirus vectors generated from the bacmid-transfected Sf9 insect cells were inoculated into Nicotiana benthamiana leaves. Confocal microscopy revealed that the gfp gene expression was high in plant leaves at 48 and 72 h after bacmid and baculovirus inoculation. Reverse transcription-PCR (RT-PCR) and fluorescence microscopy confirmed that the gfp genes under the control of Polh or CaMV35S promoters were highly expressed in plant leaves inoculated with 40 L of baculovirus solution. These results suggested that the baculovirus vector can be used to express recombinant proteins in plants. The baculovirus vector- mediated gene delivery and expression system could be used in plant biotechnology for fast and efficient production of recombinant proteins and for molecular virology studies in plants.

Engineering of baculovirus vectors for the manufacture of virion‐free biopharmaceuticals

A novel baculovirus-based protein expression strategy was developed to produce recombinant proteins in insect cells without contaminating baculovirus virions. This novel strategy greatly simplifies the downstream processing of biopharmaceuticals produced in insect cells. The formation of these virions is prevented by deletion of a baculovirus gene essential for virion formation. The deletion is trans-complemented in a transgenic insect cell line in which the baculovirus seed stock is produced. The Autographa californica multicapsid nucleopolyhedrovirus vp80 gene was selected for this purpose, as absence of VP80 prevented the formation of budded virus as well as occlusion-derived virus, while foreign gene expression was not affected. Sf9 insect cells were engineered to functionally complement the vp80 deletion in the expression vector virus during seed stock production. The trans-complemented vp80-deletion baculovirus seed produced an amount of recombinant protein similar to that produced with conventional baculovirus vectors but without contaminating virions. This novel expression method obviates the need to purify the virions away from the biopharmaceuticals.

Attempts to immunize sheep against Haemonchus contortus using a cocktail of recombinant proteases derived from the protective antigen, H-gal-GP

The objective of this study was to determine whether an antigen cocktail containing recombinantly expressed versions of most of the protective proteases of H-gal-GP, a known protective antigen from Haemonchus contortus, would confer any protection to lambs in a vaccine-challenge trial. Haemonchus contortus metalloendopeptidases, MEP1, MEP3 and MEP4, were expressed as soluble recombinant proteins in insect cells, but attempts to express the H. contortus aspartyl proteases, PEP1 and PEP2, by the same techniques were not successful. Recombinant H. contortus PEP1 was therefore expressed in Escherichia coli and refolded. Groups of sheep were immunized thrice with either native H-gal-GP, a cocktail of recombinantly expressed proteins (rMEP1, rMEP3, rMEP4 and rPep1), or adjuvant only (QuilA in PBS). All sheep were challenged with 5000 infective larvae 1 week after the final vaccination. High levels of serum antibodies that recognized H-gal-GP were detected in both the native antigen and recombinant cocktail-immunized groups by the time of challenge, but protective immunity was only observed in the group immunized with native H-gal-GP.

Efficient gene delivery into mammalian cells by recombinant baculovirus containing a hybrid cytomegalovirus promoter/Semliki Forest virus replicon

Baculovirus, which is widely utilized as an excellent tool for the production of recombinant protein in insect cells, has recently emerged as a novel and attractive gene delivery vehicle for mammalian cells. Alphavirus, such as Semliki Forest virus (SFV), has also received considerable attention for use as expression vectors because of its self-replicating property. In the present study, we investigated the characterization of recombinant baculovirus incorporating a hybrid cytomegalovirus (CMV) promoter/SFV replicon. Recombinant baculovirus containing the hybrid CMV promoter/SFV replicon was constructed. Using enhanced green fluorescence protein (EGFP) as the reporter gene, gene delivery efficiencies and the ability to express heterogenous protein in mammalian cells were evaluated. Optimal transduction conditions, including transduction temperature, time and dose, were also investigated. The obtained recombinant baculovirus, Bac-CMV/SFV-EGFP, exhibited high transduction efficiency and high-level expression of reporter protein in mammalian cells. Furthermore, this recombinant baculovirus could induce apoptosis in mammalian cells in the course of transduction, as demonstrated by the observed DNA laddering patterns and increased caspase-3 activity. The developed baculovirus vector has a high transduction efficiency and the ability to mediate foreign gene expression in mammalian cells. Taken together with its pro-apoptotic properties, this baculovirus vector may provide an alternative tool for vaccine development.