Baculovirus expression systems for recombinant protein production in insect and mammalian cells

The baculovirus vector systems has been extensively used for the expression of foreign gene products in insect and mammalian cells. New advances increase the possibilities and applications of the baculovirus expression system, which has the capability to express multiple genes simultaneously within a single infected insect cells and to use recombinant virus with mammalian cell-active expression cassettes to permit expression of recombinant proteins in mammalian cells in vitro and in vivo. Future investigations of the baculovirus expression system designed for specific target cells, can open wide variety of applications. This review summarizes the recent achievements in applications the baculovirus vector systems and optimization recombinant protein expression in both insect and mammalian cell lines.     

Baculovirus as versatile vectors for protein expression in insect and mammalian cells

Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.     

Perfusion bioreactors for the production of recombinant proteins in insect cells

Conclusion High density perfusion culture of insect cells for the production of recombinant proteins has proved to be an attractive alternative to batch and fed-batch processes. A comparison of the different production processes is summarized in Table 3. Internal membrane perfusion has a limited scale-up potential but appears to the method of choice in smaller lab-scale production systems. External membrane perfusion results in increased shear stress generated by pumping of cells and passing through microfiltration modules at high velocity. However, using optimized perfusion strategies this shear stress can be minimized such that it is tolerated by the cells. In these cases, perfusion culture has proven to be superior to batch production with respect to product yields and cell specific productivity. Although insect cells could be successfully cultivated by immobilization and perfusion in stationary bed bioreactors, this method has not yet been used in continuous processes. In fluidized bed bioreactors with continuous medium exchange cells showed reduced growth and protein production rates.For the cultivation of insect cells in batch and fedbatch processes numerous efforts have been made to optimize the culture medium in order to allow growth and production at higher cell densities. These improved media could be used in combination with a perfusion process, thus allowing substantially increased cell densities without raising the medium exchange rate. However, sufficient oxygen supply has to be guaranteed during fermentation in order to ensure optimal productivity.     

Large-scale transient expression in mammalian cells for recombinant protein production

Large-scale transient expression from mammalian cells is a new technology. Breakthroughs have been achieved for non-viral delivery methods: transfections can now be done at the 1-10 L scale with mammalian cells grown in suspension. Production of 1-20 mg/L of recombinant protein have been obtained in stirred bioreactors. Modified alphaviruses have provided a fast and efficient expression technology based on viral vectors.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and α-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and α-amylase as representatives of a simple protein and a multi-domain protein, as well as a non-glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (μmol/L) of IP was found to be higher than that of α-amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, that is, the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, (Tyo et al. submitted) in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter). For the IP there is more than 10-fold higher protein production with the CPOTud vector compared with the standard URA3-based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast.     

Oleosin fusion expression systems for the production of recombinant proteins

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals at large scale.     

A semi-automated large-scale process for the production of recombinant tagged proteins in the Baculovirus expression system

The efficient preparation of recombinant proteins at the lab-scale level is essential for drug discovery, in particular for structural biology, protein interaction studies and drug screening. The Baculovirus insect-cell expression system is one of the most widely applied and highly successful systems for production of recombinant functional proteins. However, the use of eukaryotic cells as host organisms and the multi-step protocol required for the generation of sufficient virus and protein has limited its adaptation to industrialized high-throughput operation. We have developed an integrated large-scale process for continuous and partially automated protein production in the Baculovirus system. The instrumental platform includes parallel insect-cell fermentation in 10L BioWave reactors, cell harvesting and lysis by tangential flow filtration (TFF) using two custom-made filtration units and automated purification by multi-dimensional chromatography. The use of disposable materials (bags, filters and tubing), automated cleaning cycles and column regeneration, prevent any cross-contamination between runs. The preparation of the clear cell lysate by sequential TFF takes less than 2 h and represents considerable time saving compared to standard cell harvesting and lysis by sonication and ultra-centrifugation. The process has been validated with 41 His-tagged proteins with molecular weights ranging from 20 to 160 kDa. These proteins represented several families, and included 23 members of the deubiquitinating enzyme (DUB) family. Each down-stream unit can process four proteins in less than 24 h with final yields between 1 and 100 mg, and purities between 50 and 95%.     

A serum-free medium for the culture of insect cells and production of recombinant proteins

Summary A low protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, is an effective replacement for fetal bovine serum for insect Sf-9 cells. Serum-free medium with lipid supplement and pluronic (SFM-LP) supported higher cell viability and maximum cell populations than serum-supplemented medium. No adaptation procedures are required when switching cells from serum-containing medium to SFM-LP, and growth rates remain constant during continued passages in SFM-LP. The amounts of recombinant proteins produced, which is the major use for the Sf-9 cells, are better or equal in SFM-LP compared to serum-supplemented medium. SFM-LP also supports growth of the TN-368 cell line but IPLB-SF-21AE or IZD-Mb0503 lines grow poorly in this medium.     

Baculovirus vectors for efficient gene delivery and expression in mammalian cells

Baculovirus expression vectors are extensively used for the delivery of foreign genes and expression of recombinant proteins in insect and mammalian cells. Modified baculoviruses containing mammalian promoter elements (BacMam viruses) for an efficient transient and stable transduction of diverse mammalian cells ensure a high level of heterologous protein expression both in vitro and in vivo. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus promoter, green or red fluorescent protein gene, SV40pA polyadenylation signal, and polylinker MCS were constructed for the delivery of genes encoding hepatitis C virus structural proteins into mammalian cells. In HEK293T and Huh7 cells, formation of glycoprotein complexes and HCV4ike particles was observed. A high efficiency of the baculovirus-medi-ated gene transfer and expression of the virus envelope proteins in mammalian cells was demonstrated using fluorescence, flow cytometry, and immunoblot techniques.     

Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.     

Simultaneous expression of recombinant proteins in the insect cell-baculovirus system: production of virus-like particles

The insect cell-baculovirus system (IC-BEVS) is widely used for the production of recombinant viral proteins for vaccine applications. It is especially suitable for the production of virus-like particles, which often require the simultaneous production of several recombinant proteins. Here, the available tools and process requirements for the simultaneous production of several recombinant proteins using the IC-BEVS are discussed. The production of double-layered rotavirus like particles is used as a specific example for the simultaneous production of two recombinant proteins. Methods to quantify VLP in small samples are described. The multiplicity and time of infection are presented as tools to manipulate protein concentration, and the effect on protein concentration ratios on the assembly efficiency of double-layered rotavirus like particles is discussed. It was found that not only the ratio between the recombinant proteins is determinant of VLP assembly efficiency, but also that assembly efficiency is related to the characteristics of the assembled proteins. This is the first time that kinetics of VLP production are followed during cultures, and that the assembly efficiency is quantitatively determined.     

Differential effect of exocytic SNAREs on the production of recombinant proteins in mammalian cells

Mammalian cells play a dominant role in the industrial production of biopharmaceutical proteins. However, the productivity of producer cells is often hindered by a bottleneck in the saturated secretory pathway, where a sophisticated mechanism of vesicle trafficking is mediated by numerous proteins and their complexes, among which are the cross‐kingdom conserved SNAREs [soluble NSF (N‐ethylmaleimide‐sensitive factor) receptor]. The SNAREs assemble into complexes by means of four interactive α‐helices and, thus, trigger the fusion of transport vesicles with the respective target membranes. We report that the transgenic expression of exocytic SNAREs, which control the fusion of secretory vesicles to the plasma membrane, differentially impacts the secretory capacity of HEK‐293, HeLa, and CHO‐K1 cells. While other exocytic SNAREs have no effect or a negative effect, SNAP‐23 [synaptosome‐associated protein of 23 kDa] and VAMP8 [vesicle‐associated membrane protein 8] specifically increase the production of recombinant proteins when they are ectopically and stably expressed in mammalian cells. The targeted and effective intervention in the secretory capacity of SNARE proteins is a novel engineering strategy, which could lead to the development of new therapies by increasing the production of biopharmaceutical proteins or by boosting the secretion of cell implants in cell therapy initiatives. Biotechnol. Bioeng. 2011; 108:611–620. © 2010 Wiley Periodicals, Inc.     

Improved PC1/3 production through recombinant expression in insect cells and larvae

Protein convertase 1/3 is a serine endoproteinase present in the regulated secretory pathway of endocrine and neuroendocrine cells. It is responsible for the processing of numerous prohormones and proneuropeptides into their biologically active moieties, often following cleavage at pairs of basic residues. The determination of its three-dimensional structure, as well as the understanding of its enzymatic properties, would greatly benefit from the production and availability of large amounts of recombinant enzyme. We report herein improvements in the production of PC1/3 by expressing recombinant mutated forms in both insect cells (Spodoptera frugiperda, Sf9 cells) and larvae (Trichoplusia ni commonly referred to as cabbage looper). On one hand, we deleted the last 135 COOH-terminal residues of mPC1/3 and, on the other hand, we replaced the signal peptide of mPC1/3 by the viral glycoprotein gp67 signal peptide. These modifications were shown to improve markedly (up to 125%) the secretion into the Sf9 cells medium and the amount of enzymatic activity recovered when compared to the original vector. Moreover, intracoelemic injection of the vectors into insect larvae led to the production and purification of enzymatically active enzyme at a level of 30 microg/larva in the case of mPC1/3 and to the production of a high amount of another enzymatically active convertase, PC7. The optimal viral titer for infection of larvae was determined to be 10(6)pfu/ml. Taking into account the purification protocol combined with the ease and efficiency of using larvae, it should now be possible to meet the needs for biochemical and structural studies.     

Using Vaccinia's innate ability to introduce DNA into mammalian cells for production of recombinant proteins

Production of recombinant protein in mammalian cells is time-consuming, labor-intensive and costly. While seeking to overcome these limitations, we discovered that Vaccinia virus has the innate ability to transfer exogenous plasmid DNA into mammalian cells during the infection process. Parameters influencing the efficiency of this event were characterized and a quick, simple and inexpensive way to produce eukaryotic proteins was established.     

A high-level expression vector containing selectable marker for continuous production of recombinant protein in insect cells

Insect cell expression systems are widely used to produce active recombinant proteins. Here, we have developed a high-level expression vector containing a selectable marker for continuous production of recombinant proteins in insect cells. The plasmid, pXIHAbla, developed in this study, established a polyclonal cell line 8 days shorter than pXINSECT-DEST38 and pBmAneo. In addition, pXIHAbla exhibited an approximately fivefold higher average enhanced GFP expression level and approximately a twofold higher bionanocapsule secretion level than pXINSECT-DEST38. Using this plasmid, insect cells that highly express active proteins have been easily established.     

Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells.     

Recombinant baculoviruses as expression vectors for insect and mammalian cells.

Baculovirus expression vectors are widely used for expressing heterologous proteins in cultured insect cells. Recent advances include further development of the system for production of multi-subunit protein complexes, co-expression of protein-modifying enzymes to improve heterologous protein production, and additional applications of baculovirus display technology. The application of modified baculovirus vectors for gene expression in mammalian cells continues to expand.