Large-scale Production Of Recombinant Proteins Research Articles

Two-compartment bioreactor as a scale-down model to study the effect of glucose overflow and anaerobiosis on large-scale recombinant protein production processes

Background In high-cell density fermentations the host cells are often subjects of transient changes in microenvironment around them. This is true especially in large-scale bioreactors. The changes can be for example substrate gradient, differences in oxygen availability and pH variations. Our aim is to obtain more information about physiological changes of E.coli W3110 and its recombinant variants in such conditions for better understanding of the bottlenecks in recombinant protein production processes.

Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript

The efficient scale-up of recombinant protein production in insect-cell bioreactors using baculovirus expression vectors is hampered by reductions in yield with increasing viral passage, the so-called passage effect. This phenomenon is characterized by the generation and subsequent accumulation of defective interfering baculoviruses (DIs), which interfere with the replication of genomically intact virus. A novel baculovirus expression vector is presented equipped with a bicistronic expression cassette that allows the simultaneous expression of the recombinant gene (GFP, first cistron) and an essential baculovirus gene (GP64, second cistron) from a single messenger RNA (mRNA). The translation of GP64 is mediated by an internal ribosome entry site (IRES) element from Rhopalosiphum padi virus (RhPV) while the native GP64 gene is deleted. In this way, a dominant selection pressure is placed on the entire bicistronic mRNA and hence on the maintenance of the foreign gene. The bicistronic expression vector was superior to the control baculovirus vector in that GFP expression remained at much higher levels upon continued virus passage. The versatility of this stabilized vector was demonstrated by its ability to propagate in a number of cell lines including Sf21, Sf9 and High Five cells. This novel baculovirus vector is especially valuable for large-scale recombinant protein production in insect-cell bioreactors where the number of viral passages is high.

F4 (K88) fimbrial adhesin FaeG expressed in alfalfa reduces F4+ enterotoxigenic Escherichia coli excretion in weaned piglets

Transgenic plants are attractive bioreactors to large-scale production of recombinant proteins because of their relatively low cost. This study reports for the first time the use of transgenic plants to reduce enterotoxigenic Escherichia coli (ETEC) excretion in its natural host species. The DNA sequence encoding the major subunit and adhesin FaeG of F4+ ETEC was transformed into edible alfalfa plants. Targeting of FaeG production to chloroplasts led to FaeG levels of up to 1% of the total soluble protein fraction of the transgenic alfalfa. Recombinant plant-produced FaeG (pFaeG) remained stable for 2 years when the plant material was dried and stored at room temperature. Intragastric immunization of piglets with pFaeG induced a weak F4-specific humoral response. Co-administration of pFaeG and the mucosal adjuvant cholera toxin (CT) enhanced the immune response against FaeG, reflected a better induction of an F4-specific immune response. In addition, the intragastric co-administration of CT with pFaeG significantly reduced F4+ E. coli excretion following F4+ ETEC challenge as compared with pigs that had received nontransgenic plant material. In conclusion, transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against F4+ ETEC infections.

Advances in Fabrication of Calcium Phosphate Nano-composites for Smart Delivery of DNA and RNA to Mammalian Cells

Over the past 30 years, tremendous efforts have been made for the development of a safe, costeffective and efficient device for nucleic acid delivery to mammalian cells. The approach has great contribution to analysis of gene expression, function and regulation and production of numerous recombinant therapeutic proteins, as well as bright prospects for human gene therapy. Of the existing techniques either based on synthetic material (lipid, peptide, polymer, inorganic crystal) or natural one (virus), calcium phosphate (CaP) precipitation is the most simplified and the least expensive and thus widely used for basic research and largescale production of recombinant proteins. In addition, implementation of CaP precipitates in gene therapy purposes has recently been started. So this the right time to discuss the current progress in understanding the growth kinetics and physico-chemical properties of CaP precipitates, as well as the strategies undertaken so far to modulate the growth profiling which seriously affects the sizes of the precipitates - a crucial factor for effective delivery of DNA or RNA to the cells through endocytosis. Among the approaches in nano-apatite technology, magnesium-inspired nano-apatite formulation appears to have the tremendous potential for transfecting mammalian cells considering the flexibility and simplicity in regulating crystal growth kinetics at the molecular level and eventual remarkable efficiency for both delivery and expression of an exogenous gene. Keywords: calcium phosphate, magnesium, nano-particles, crystal growth, delivery, dna, expression, sirna, gene silencing

870. Transfection of Mammalian Cells Using Linear Polyethylenimine Is a Simple and Effective Means of Producing Recombinant AAV

We have developed a very simple protocol to transfect mammalian cells with linear polyethylenimine (PEI). Linear PEI has been used to transfect nucleic acids into a wide variety of mammalian cells both in cell culture and in vivo. Almost all of these reports have used commercially available preparations of PEI. Our linear PEI protocol is as efficient as commercial reagents in the transfection of HEK293 and HeLa cells, but at only a fraction of the cost. Our protocol should be useful for many different applications. It should be particularly useful for large-scale production of recombinant proteins and viruses requiring transfection of mammalian cells in large batches. We have used this protocol to produce recombinant AAV, which requires transfection of cells with three plasmids simultaneously. One plasmid contains the recombinant AAV genome. The second plasmid expresses the AAV rep and cap genes. The third plasmid expresses several adenovirus genes that are required for AAV replication. We have assessed production of a recombinant AAV that expresses GFP both by quantitative PCR and by transduction of HeLa cells.

The CELO adenovirus Gam1 protein enhances transient and stable recombinant protein expression in Chinese hamster ovary cells

The Gam1 protein of the avian CELO adenovirus activates transcription through inhibition of histone deacetylase 1 (HDAC1). We investigated the effect of Gam1 on both transient and stable transgene expression in Chinese hamster ovary (CHO) cells, one of the most commonly used mammalian hosts for the large-scale production of recombinant proteins. Transient expression of Gam1 increased reporter protein levels up to 4-fold in suspension cultures of CHO DG44 cells co-transfected with a reporter gene and up to 20-fold in recombinant CHO DG44-derived cell lines. The highest levels of activation were observed when the transgene was under the control of the human cytomegalovirus (HCMV) immediate early promoter/enhancer. Increases in recombinant protein expression in the presence of Gam1 were not accompanied by an enhancement of cell growth or viability. We conclude that Gam1 may serve as a useful genetic tool for increasing recombinant protein expression in CHO DG44 cells.

Opportunities for recombinant antigen and antibody expression in transgenic plants—technology assessment

Plants are now gaining widespread acceptance as a general platform for the large-scale production of recombinant proteins. The principle has been demonstrated by the success of a diverse repertoire of proteins, with therapeutic molecules showing the most potential for added value. Over the past 10 years, several efficient plant-based expression systems have emerged. However, a number of issues remain to be addressed before plant bioreactors can be accepted and adopted widely in preference to the established microbial and mammalian platforms. Overcoming bottlenecks imposed by low yields, poor and inconsistent product quality and difficulties with downstream processing are the most important goals for researchers working in this field. The achievement of these goals is conditional on the development of extraction and processing steps that comply with GMP standards, including extensive quality assurance and control procedures. Such rigorous and validated standards should be combined with measures applied earlier in production to ensure product sustainability and quality, such as the use of master seed banking procedures. Moreover, there are several further challenges concerning topics of environmental impact, biosafety and risk assessment, which reflect the release of transgenic plants, as well the safety of the plant-derived products themselves. We are facing a growing demand for protein diagnostics and therapeutics, but lack the capacity to meet those demands using established facilities. A shift to plant bioreactors may, therefore, become necessary within the next few years, making it more imperative that the technical and regulatory limitations are addressed and solved. The production of pharmaceutical proteins in plants will only realize its huge potential if the products are provided at consistent high quality levels, allowing the delivery of clinical grade proteins that will gain regulatory approval and which can be used routinely in clinical trials.

The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P. pastoris

The methylotrophic yeast Pichia pastoris is a popular host for the production of a variety of recombinant proteins . We describe the use of a novel selectable marker , the P. pastoris formaldehyde dehydrogenase gene ( FLD1 ) for DNA-mediated transformations of this yeast. The product of the FLD1 gene (Fld1p) is required for growth of P. pastoris on methanol as a carbon source and methylamine as a nitrogen source. In both these C 1 pathways, Fld1p oxidizes formaldehyde to formate, which is subsequently further oxidized by a second dehydrogenase to carbon dioxide. We show that the FLD1 gene can be used as a marker in transformations of a P. pastoris fld1 host by selection on plates containing methylamine. Furthermore, we demonstrate that populations of these transformants can be enriched for strains that receive multiple copies of an FLD1 -based vector by their increased resistance to formaldehyde. We provide the FLD1 selection system in a set of P. pastoris expression vectors that are composed almost entirely of P. pastoris DNA (except for the recombinant gene) and are devoid of antibiotic resistance genes or other sequences of bacterial origin. The vectors are useful for the selection of strains containing multiple copies of an expression vector and may be ideal for certain large-scale recombinant protein production processes where strains containing non- P. pastoris DNA sequences , particularly bacterial antibiotic resistance genes and replication origins , are considered a potential biological hazard to be avoided.

The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P. pastoris.

The methylotrophic yeast Pichia pastoris is a popular host for the production of a variety of recombinant proteins. We describe the use of a novel selectable marker, the P. pastoris formaldehyde dehydrogenase gene (FLD1) for DNA-mediated transformations of this yeast. The product of the FLD1 gene (Fld1p) is required for growth of P. pastoris on methanol as a carbon source and methylamine as a nitrogen source. In both these C(1) pathways, Fld1p oxidizes formaldehyde to formate, which is subsequently further oxidized by a second dehydrogenase to carbon dioxide. We show that the FLD1 gene can be used as a marker in transformations of a P. pastoris fld1 host by selection on plates containing methylamine. Furthermore, we demonstrate that populations of these transformants can be enriched for strains that receive multiple copies of an FLD1-based vector by their increased resistance to formaldehyde. We provide the FLD1 selection system in a set of P. pastoris expression vectors that are composed almost entirely of P. pastoris DNA (except for the recombinant gene) and are devoid of antibiotic resistance genes or other sequences of bacterial origin. The vectors are useful for the selection of strains containing multiple copies of an expression vector and may be ideal for certain large-scale recombinant protein production processes where strains containing non-P. pastoris DNA sequences, particularly bacterial antibiotic resistance genes and replication origins, are considered a potential biological hazard to be avoided.

Functional expression and direct visualization of the human α 2B-adrenergic receptor and α 2B-AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

The α 2B-adrenergic receptor (α 2B-AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the α 2B-AR. The receptor fusions α 2B-AR–eGFP and CAP–α 2B-AR expressed in CHO-K1 cells generated B max values of 176 and 122 pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, α 2B-AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.

Recombinant protein production by the baculovirus-insect cell system in Basal media without serum supplementation.

The production of beta-galactosidase by Sf9 cells infected with recombinant Autographa californica nucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the beta-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted beta-galactosidase production, their removal by medium replacement on post-infection day 1 gave a beta-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing beta-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.

Semliki Forest virus vectors for large-scale production of recombinant proteins.

AbstractRecombinant protein expression is one of the basic elements of modern molecular biology. The completion of the sequencing of the human genome has, in fact, accelerated the need of rapid and efficient expression systems. Extensive efforts have been dedicated to the development of expression systems for bacterial, yeast, insect, and mammalian cells. The reason for using mammalian expression systems is obviously the correct posttranslational modification capacity and the most authentic environment for production of therapeutically interesting proteins. Today, it is also important that large quantities of recombinant proteins be produced and subjected to purification and structural studies to obtain high-resolution structures to aid rational drug design.KeywordsSuspension CultureVirus StockRecombinant Protein ExpressionBaby Hamster KidneySemliki Forest VirusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Control of recombinant human endostatin production in fed-batch cultures of Pichia pastoris using the methanol feeding rate

Endostatin is a 20 kDa carboxyl-terminal fragment of collagen XVIII that strongly inhibits angiogenesis and tumor growth. The methylotrophic yeast, Pichia pastoris, is a robust expression system that can be used to study methods to improve the yields of rhEndostatin. We expressed rhEndostatin in P. pastoris under the control of the alcohol oxidase 1 (aox 1) promoter (Mut+ phenotype) as a model, and used a cell biomass of about 50 g l−1 dry cell wt as a starting point for the induction phase and varied the methanol feed rate at 8 ml l−1 h−1, 11 ml l−1 h−1 and 15 ml l−1 h−1. While the cell growth rate was proportional to the rate of methanol delivery, protein production rate was not. These findings could be used to guide parameters for large-scale production of recombinant proteins in the P. pastoris system.

Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli.

Escherichia coli, a venerable workhorse for biochemical and genetic studies and for the large-scale production of recombinant proteins, is one of the most intensively studied of all organisms. The natural habitat of E. coli is the gastrointestinal tract of warm-blooded animals, and in humans, this species is the most common facultative anaerobe in the gut. Although most strains exist as harmless symbionts, there are many pathogenic E. coli strains that can cause a variety of diseases in animals and humans. In addition, from an evolutionary perspective, strains of the genus Shigella are so closely related phylogenetically that they are included in the group of organisms recognized as E. coli (1, 2). Pathogenic E. coli strains differ from those that predominate in the enteric flora of healthy individuals in that they are more likely to express virulence factors — molecules directly involved in pathogenesis but ancillary to normal metabolic functions. Expression of these virulence factors disrupts the normal host physiology and elicits disease. In addition to their role in disease processes, virulence factors presumably enable the pathogens to exploit their hosts in ways unavailable to commensal strains, and thus to spread and persist in the bacterial community. It is a mistake to think of E. coli as a homogenous species. Most genes, even those encoding conserved metabolic functions, are polymorphic, with multiple alleles found among different isolates (1). The composition of the genome of E. coli is also highly dynamic. The fully sequenced genome of the laboratory K-12 strain, whose derivatives have served an indispensable role in the laboratories of countless scientists, shows evidence of tremendous plasticity (3). It has been estimated that the K-12 lineage has experienced more than 200 lateral transfer events since it diverged from Salmonella about 100 million years ago and that 18% of its contemporary genes were obtained horizontally from other species (4). Such fluid gain and loss of genetic material are also seen in the recent comparison of the genomic sequence of a pathogenic E. coli O157:H7 with the K-12 genome. Approximately 4.1 million base pairs of “backbone” sequences are conserved between the genomes, but these stretches are punctuated by hundreds of sequences present in one strain but not in the other. The pathogenic strain contains 1.34 million base pairs of lineage-specific DNA that includes 1,387 new genes; some of these have been implicated in virulence, but many have no known function (5). The virulence factors that distinguish the various E. coli pathotypes were acquired from numerous sources, including plasmids, bacteriophages, and the genomes of other bacteria. Pathogenicity islands, relatively large (>10 kb) genetic elements that encode virulence factors and are found specifically in the genomes of pathogenic strains, frequently have base compositions that differ drastically from that of the content of the rest of the E. coli genome, indicating that they were acquired from another species. Here, we explore some of the known virulence factors that contribute to the heterogeneity of E. coli strains, and we review what is known regarding the origin and distribution of these factors.

Protein Expression in the Baculovirus System

Insect cell-recombinant baculovirus co-cultures offer a protein production system that complements microbial systems by providing recombinant proteins in soluble form and with most post-translational modifications. Moreover, the large size of the viral genome enables cloning of large segments of DNA and consequent expression of complex protein aggregates. This unit describes methods associated with the large-scale production of recombinant proteins in the baculovirus expression system. A method for large-scale production of viral stocks is described and methods for titration of virus are provided (a plaque assay and an end-point assay). Once viral stocks have been prepared and titered, a protocol for testing the virus in small-scale cultures is provided to determine the kinetics of expression, which allows evaluation of various cell culture and infection conditions aimed at developing optimal levels of protein production (e.g., comparisons of different host cell lines, media, and environmental parameters). Support protocols provide instructions for preparing culture samples for protein analysis by SDS-PAGE and discuss analytical methods for monitoring nutrient levels in cell culture fluids. Once optimal process parameters are identified, protocols describe production of the target protein on a large scale in fermentors using either regular batch production in bioreactors or a fed-batch procedure of production in perfusion cultures. Techniques for harvesting cultures from bioreactors are also provided.

Direct refolding of recombinant human growth differentiation factor 5 for large-scale production process

An efficient downstream process for the production of recombinant human growth differentiation factor 5 (rhGDF5) has been developed for industrial application utilizing a novel “direct refolding” method. In this method, the starting material is an inclusion body produced in Escherichia coli, and the critical step is the direct refolding step that follows directly after solubilization of the inclusion body. rhGDF5 can be refolded at a markedly high concentration of 2.4 mg·ml −1, which is 24 times that hitherto achieved by the proteins of the TGF-β superfamily. The refolding yield is 63%, and after purification by diafiltration, isoelectric precipitation and reverse-phase chromatography, the final purification yield is 20% with purity higher than 99%. The yield is more than twice that of a conventionally established process having three chromatography steps and the purity is equivalent. The first pilot-scale trial shows a refolding yield of 51% and a final yield of 11%. The final yield is 1.4 times that of the conventional process, and further optimization at pilot-scale is expected to bring this figure up to or above that of laboratory-scale. As a result, the calculated production cost of rhGDF5 has been reduced dramatically. This type of efficient and simple process is beneficial particularly in the large-scale production of recombinant proteins in which high yield and quality are required.

The use of bi-cistronic transfer vectors for the baculovirus expression system

In this communication, we describe the construction of bi-cistronic transfer vectors for the baculovirus expression system (BVES), which are advantageous over the existing vectors. The new vectors provide a simple way to isolate recombinant viruses. More specifically, the gene of interest and the reporter gene luciferase (LUC), constitute the first and second cistrons, respectively, of the same transcript. Therefore, the LUC activity measured during infection of such a bi-cistronic virus, permits an on-line estimation of the recombinant protein level, a very useful feature for large-scale production of recombinant proteins. To achieve expression of the second cistron, the internal ribosome entry site (IRES) element of the encephalomyocarditis virus (EMCV) was employed. However, this element, which is highly efficient in mammalian systems, did not promote efficient internal translation of the second cistron in various insect cells lines originating from different insect species. The lack of efficient internal translation was not due to baculovirus propagation since the same phenomenon was also observed in a viral-free expression system. It seems that a component essential for efficient EMCV IRES activity is either missing or present in limiting amount in insect cells or not compatible. Nevertheless, LUC placed downstream to the IRES element, or immediately downstream to the first cistron, was expressed to a level that enabled the biotechnological application it was designed for.

Production of recombinant proteins in plant root exudates.

The large-scale production of recombinant proteins in plants is limited by relatively low yields and difficulties in extraction and purification. These problems were addressed by engineering tobacco plants to continuously secrete recombinant proteins from their roots into a simple hydroponic medium. Three heterologous proteins of diverse origins (green fluorescent protein of jellyfish, human placental alkaline phosphatase [SEAP], and bacterial xylanase) were produced using the root secretion method (rhizosecretion). Protein secretion was dependent on the presence of the endoplasmic reticulum signal peptide fused to the recombinant protein sequence. All three secreted proteins retained their biological activity and, as shown for SEAP, accumulated in much higher amounts in the medium than in the root tissue.

Methods for Baculovirus Amplification - Application to Large Scale Recombinant Protein Production in Insect Cells

Baculovirus amplification in one insect cell line Spodoptera frugiperda (Sf21) and subsequent recombinant protein production in another cell line, Trichoplusia ni, has been achieved within a single bioreactor. The advantages of this single bioreactor configuration include minimization of the virus volumes and titres required for large scale protein expression experiments as well as optimization of the infection process itself.

Baculovirus multigene expression vectors and their use for understanding the assembly process of architecturally complex virus particles

The baculovirus expression vector is a eukaryotic DNA viral vector for the cloning and expression of foreign genes in cultured lepidopteran insect cells and insects. It has become an important tool for the large-scale production of recombinant proteins for a variety of applications including the structure-function analysis of genes and their gene products. We have developed a number of baculovirus multigene expression vectors and utilized these to understand the assembly process of multicomponent capsid structures of large viruses such as bluetongue virus (BTV), a member of the Orbivirus genus within the family Reoviridae. BTV is some 810 Å in diameter and comprised of two protein shells containing four major proteins, VP2, VP5, VP7 and VP3, surrounding a genome of ten double-stranded RNA segments and three minor proteins (VP2, VP4 and VP6). BTV is the etiological agent of a sheep disease that is sometimes fatal in certain parts of the world (e.g., Africa, Asia, and the Americas). Using baculovirus multigene vectors, we have co-expressed various combinations of BTV genes in insect cells and produced structures that mimic the various stages of BTV assembly. For example, co-expressed VP3 and VP7 form BTV core-like particles, while co-expressed VP2, VP5, VP7 and VP3 form BTV virus-like particles. Using deletion, point and domain switching analyses of each protein, we have been able to identify certain sequences in the VP7 and VP3 proteins that are essential for the assembly of core-like particles. These expression and biochemical studies have been complemented by collaboration studies using cryoelectron microscopy and image processing analyses to provide the three-dimensional structure of the expressed particles. In addition and with other associates, we have used X-ray crystallography of VP7 to deduce its atomic structure. Extensive studies on the immune responses elicited by these self-assembled particles, and chimeric derivatives involving various foreign antigens, have been carried out. Finally, using as little as 10 μg of the self-assembled virus-like particles, we have shown that they can confer long-lasting protection in sheep against BTV.