Large-scale Protein Production Research Articles

Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells

Hydrophobins play an important role in binding and assembly of fungal surface structures as well as in medium–air interactions. These, hydrophobic properties provide interesting possibilities when purification of macromolecules is concerned. In aqueous micellar two-phase systems, based on surfactants, the water soluble hydrophobins are concentrated inside micellar structures and, thus, distributed to defined aqueous phases. This, one-step purification is attractive particularly when large-scale production of recombinant proteins is concerned. In the present study the hydrophobin HFBI of Trichoderma reesei was expressed as an N-terminal fusion with chicken avidin in baculovirus infected insect cells. The intracellular distribution of the recombinant fusion construct was analyzed by confocal microscopy and the protein subsequently purified from cytoplasmic extracts in an aqueous micellar two-phase system by using a non-ionic surfactant. The results show that hydrophobin and an avidin fusion thereof were efficiently expressed in insect cells and that these hydrophobic proteins could be efficiently purified from these cells in one-step by adopting an aqueous micellar two-phase system.

PERSISTENCE AND DEGRADATION OF MAIZE-EXPRESSED VACCINE PROTEIN, ESCHERICHIA COLI HEAT-LABILE ENTEROTOXIN SUBUNIT B, IN SOIL AND WATER

Transgenic plants represent an innovative platform for the cost-effective large-scale production of various pharmaceutical proteins. The eventual open-field production of plant-made pharmaceuticals (PMPs) requires risk assessment to determine the potential for harm to the surrounding ecosystem. In the present study, the environmental persistence of a transgenic maize-expressed antigen, Escherichia coli heat-labile enterotoxin subunit B (LTB), was studied under laboratory conditions. To semiquantitatively monitor the persistence of LTB in soil, extraction with a high-salt, high-pH extraction buffer was optimized using the closely homologous Vibrio cholerae enterotoxin subunit B (CTB) as a test substance. The time to dissipation of 50% (DT50) of the extractable fraction of maize-expressed LTB was 4 to 15 d in pond water and 35 to 90 d in soils. Both extraction efficacy and persistence were strongly affected by the matrix type and incubation conditions. In contrast with maize-expressed LTB, the DT50 for bacterially produced LTB and CTB was less than 4 d both in pond water and soil. Although maize-expressed LTB was more stable than bacterially produced analogue, its dissipation was governed by an initial lag, which could be attributed to release from the plant material, followed by rapid decline.

Flow cytometry for the rapid detection of bacteria in cell culture production medium

The rapid and sensitive detection of microbial contaminants is critical for timely contamination investigations and monitoring of process control for in vitro protein production systems. A flow cytometric method was developed to monitor two types of cell culture media used in large scale protein production. The process used a DNA dye, thiazole orange, which binds to nucleic acids of viable and nonviable organisms. To ensure a representative sample was tested and to enhance the detection limit, a concentration step before staining and analysis included a double centrifugation. In addition, a washing step was included to eliminate background fluorescence caused by material in the media formulations. The staining and analysis of concentrated and washed samples takes approximately 0.5 h and provides objective results. The feasibility of the method was demonstrated by spiking sterile media with six bacterial species that represent the most commonly encountered bacterial contaminants. In addition to the bacterial spiking study, 164 lots of large scale production medium were tested by the flow cytometric method in parallel with conventional culture using Trypticase Soy Broth (TSB). In the bacterial spiking study, the concentration method increased the microbial titer by 2 logs. The detection limit of organisms within the medium was determined to be 1 CFU/ml. There was 100% correlation between the flow cytometric method and the standard aerobic plate count method using Trypticase Soy Agar. In the parallel study on 164 lots of large scale production media, the flow cytometric method was compared against culture in TSB. The sensitivity of the method was determined to be 100%, the specificity was 99.4%, the positive predictive value was 83%, and the negative predictive value was 100%. To reduce reporting of false positives, an initial positive result should be confirmed by an additional sample. The analytic sensitivity, specificity, and objectivity of this flow cytometric method indicate this method is valuable in the monitoring of production media for microbial contamination.

Conquering isoleucine auxotrophy of Escherichia coli BLR(DE3) to recombinantly produce spider silk proteins in minimal media

Large-scale production of recombinant spider silk proteins is a long-term goal for their industrial use. Therefore, we have recently developed a process for bacterial production. Due to a highly repetitive gene sequence of spider silks, the host strain E. coli BLR(DE3) was employed since it shows no homologue recombination. Although perfectly suited for production in full media, the BLR strain does not grow in cost-effective minimal media, indicating a previously not reported L: -isoleucine auxotrophy. We provide evidence that mutated threonine deaminase is likely responsible for the detected auxotrophy of BLR.

High-Level Expression of Fusion Protein Containing 10 Tandem Repeated GLP-1 Analogs in YeastPichia pastorisand Its Biological Activity in a Diabetic Rat Model

Glucagon-like peptide-1 (GLP-1), an incretin secreted by intestinal L-cells, can effectively lower blood glucose levels in patients with diabetes. A fusion gene, consisting of 10 tandem repeated GLP-1 analog genes, was expressed at a high level in the yeast Pichia pastoris. SDS polyacrylamide gel electrophoresis (SDS-PAGE), and Western Blotting results showed that fusion protein migrated as a single protein band with a molecular weight of 36 kDa. A biological activity test showed that the GLP-1 analog could significantly lower the level of serum glucose when GLP-1 purified analog was injected into diabetic rats. A potential strategy for large-scale production of fusion protein containing the 10 GLP-1 analogs as discovered, and a single GLP-1 analog was obtained from fusion protein digested with trypsin. This should be inspired foreign expression of medicinal short peptides and be valuable in further research on GLP-1 analog drugs in the treatment of diabetes mellitus.

Viral vectors for the expression of proteins in plants

The use of plant viral vectors for the transient expression of heterologous proteins offers a useful tool for the large-scale production of proteins of industrial importance, such as antibodies and vaccine antigens. In recent years, advances have been made both in the development of first-generation vectors (that employ the 'full virus') and second-generation ('deconstructed virus') vectors. For example, vectors based around the 'full virus' strategy can now be used to express long polypeptides (at least 140 amino acids long) as fusions to the coat protein. In addition, a new generation of vectors was engineered to have a reactogenic amino acid exposed on the surface of the virus, allowing easy chemical conjugation of (separately produced) proteins to the viral surface. This approach is being used to develop new vaccines in the form of antigens coupled to a plant viral surface. Prototypes of industrial processes that require high-yield production, rapid scale-up, and fast manufacturing have been recently developed using the 'deconstructed virus' approach (magnifection). This process, which relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA replicons to plant cells, has been shown to work with numerous proteins, including full immunoglobulin G antibodies. Other advances in this area have looked at the development of inducible viral systems and the use of viral vectors to produce nanoscale materials for modular assembly.

A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag

We describe a generic protocol for the overproduction and purification of recombinant proteins in Escherichia coli. The strategy utilizes a dual His6-maltose binding protein (HisMBP) affinity tag that can be removed from the target protein by digestion of the fusion protein at a designed site by tobacco etch virus protease. The MBP moiety serves to enhance the solubility and promote the proper folding of its fusion partners, and the polyhistidine tag facilitates its purification to homogeneity. This protocol is divided into three stages, each of which takes approximately 1 week to complete: (i) construction of a HisMBP fusion vector; (ii) a pilot experiment to assess the yield and solubility of the target protein; and (iii) the large-scale production and purification of the target protein.

Combined approach of NMR and chemometrics for screening peptones used in the cell culture medium for the production of a recombinant therapeutic protein

Soy peptones or soy hydrolysates are widely used as key medium additives in serum-free cell culture processes for industrial production of therapeutic recombinant proteins. The heterogeneous nature of these vegetable-derived materials can lead to substantial lot- to-lot variability in cell culture processes. In this study, we demonstrated the feasibility of nuclear magnetic resonance (NMR) spectroscopy in combination with chemometrics in rapid screening peptone lots in order to optimize efficiency and consistency of large-scale protein production. This report is the first that shows a correlation between the intrinsic NMR spectral characteristics of complex heterogeneous materials and product titer using chemometrics.

Persistence and degradation of maize-expressed vaccine protein, Escherichia coli heat-labile enterotoxin subunit B, in soil and water

Transgenic plants represent an innovative platform for the cost-effective large-scale production of various pharmaceutical proteins. The eventual open-field production of plant-made pharmaceuticals (PMPs) requires risk assessment to determine the potential for harm to the surrounding ecosystem. In the present study, the environmental persistence of a transgenic maize-expressed antigen, Escherichia coli heat-labile enterotoxin subunit B (LTB), was studied under laboratory conditions. To semiquantitatively monitor the persistence of LTB in soil, extraction with a high-salt, high-pH extraction buffer was optimized using the closely homologous Vibrio cholerae enterotoxin subunit B (CTB) as a test substance. The time to dissipation of 50% (DT50) of the extractable fraction of maize-expressed LTB was 4 to 15 d in pond water and 35 to 90 d in soils. Both extraction efficacy and persistence were strongly affected by the matrix type and incubation conditions. In contrast with maize-expressed LTB, the DT50 for bacterially produced LTB and CTB was less than 4 d both in pond water and soil. Although maize-expressed LTB was more stable than bacterially produced analogue, its dissipation was governed by an initial lag, which could be attributed to release from the plant material, followed by rapid decline.

High‐Level Recombinant Protein Production in CHO Cells Using an Adenoviral Vector and the Cumate Gene‐Switch

To facilitate and accelerate the production of eukaryotic proteins with correct post-translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO-cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene-switch transcription system. This cell line is adapted to suspension culture and can grow in serum-free and protein-free medium. To increase the transduction level of AdVs, we have also generated a cell line (CHO-cTA-CAR) that expresses additional amounts of the coxackievirus and adenovirus receptor (CAR) on its surface. Recombinant protein production was tested using an AdV carrying the secreted alkaline phosphatase (SEAP) under the control of the CR5 promoter, which is strongly and specifically activated by binding to cTA. The SEAP expression was linked to the expression of the green fluorescent protein (GFP) through an internal ribosome entry site (IRES) to facilitate titration of the AdV. We monitored SEAP expression on a daily basis for 9 days after transduction of CHO-cTA and CHO-cTA-CAR using different quantities of AdVs at 37 and 30 degrees C. Incubation at the latter temperature increased the production of SEAP at least 10-fold, and the presence of CAR increased the transduction level of the AdV. Maximum SEAP production (63 mg/L) was achieved at 6-7 days post-infection at 30 degrees C by transducing CHO-cTA-CAR with 500 infectious particles/cell. Because numerous AdVs can now be generated within a few weeks and large-scale production of AdVs is now a routine procedure, this system could be used to produce rapidly milligram quantities of a battery of recombinant proteins as well as for large-scale protein production.

High-level expression of human lactoferrin in the milk of goats by using replication-defective adenoviral vectors

The expression of human lactoferrin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce lactorferrin in the milk of transgenic animals resulted in very high cost and uncertain results. In this paper, we have directly infused replication-defective adenovirus encoding human lactoferrin cDNA into the mammary gland of goats via the teat canal. In this way, we obtained a high level of expressed human lactoferrin up to 2 g/L in the milk of goats. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS–PAGE and Western blotting. A ∼80-kDa protein was visualized after viral vector infection. Our results demonstrate that intraductal injection of recombinant replication-defective adenovirus vectors may provide a very useful tool for large-scale production of recombinant proteins of biopharmaceutical interest.

Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli

In our previous studies, the recombinant gonadotropin-releasing hormone (GnRH) peptide was constructed into a T7 RNA polymerase-based expression system. The recombinant gene encoding GnRH3-hinge-MVP, which contained three repeated GnRH units, a fragment of hinge region (225-232/225'-232'), and a T cell epitope of measles virus protein, was cloned into Escherichia coli BL21 harboring pED-GnRH3. The high activity of T7 RNA polymerase could make the expression system very powerful for high-level expression of the recombinant protein. However, during the large-scale production of recombinant protein, the productivity of the fermentation process was directly affected by many factors, such as plasmid stability, protein production, and culture conditions. In this study, we studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, the time of induction by lactose, and the number of successive cultures. The results indicate that the plasmid instability may be caused by a loss of plasmid rather than structural change. However, to go down to future generations, engineered bacteria have the stability of plasmid and protein yield to a large extent. The amount of the fusion protein was also up to 40% of the total cell protein after the 50th generation. These data would be useful for the industrial production of the recombinant GnRH vaccine.

Multiplexed expression and screening for recombinant protein production in mammalian cells.

BackgroundA 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.ResultsA nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale.ConclusionThe combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful for applications such as the generation of receptor protein microarrays.

Comparison of several Nicotiana species as hosts for high‐scale Agrobacterium‐mediated transient expression

Agrobacterium-mediated transient expression may be regarded as a promising method for inexpensive large-scale production of recombinant proteins. We optimized the protocol of transient expression in Nicotiana benthamiana and compared six Australian species of Nicotiana as hosts for transient expression. The transient expression of GFP under 35S CaMV promoter was observed in all species tested, although the GFP content in leaves of N. benthamiana, N. exigua, and N. excelsior was significantly higher (3.8, 3.7, and 2.0% TSP, respectively). Usage of viral-based expression system resulted in considerable increase of GFP accumulation in N. excelsior and N. benthamiana (63.5 and 16.2% TSP, respectively). We displayed that N. excelsior has the best characteristics in regard to biomass yield as well as GFP accumulation level for both types of the expression cassettes tested.

Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors

We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine (PEI). Our linear PEI protocol is as effective as commercial reagents in the transfection of HeLa cells and XDC293 cells, a derivative of HEK293 cells, but at a fraction of the cost. Greater than 90% of XDC293 cells and 98% of HeLa cells transfected using our method were positive for EGFP expression as determined by flow cytometery. Our protocol should be useful for many different applications such as large-scale production of recombinant protein and viruses, which requires transient transfection of mammalian cells in large batches. We have used this protocol to produce recombinant adeno-associated virus (AAV) in XDC293 cells and in HeLa cells. This requires transient expression of three adenovirus gene-products (E2A, E4orf6, and VA RNAs) as well as the AAV replication (Rep78, Rep68, Rep52, and Rep40) and capsid (VP1, VP2, and VP3) proteins. Production of a recombinant AAV that expresses green fluorescent protein was assessed by quantitative PCR and by transduction of HeLa cells. Linear PEI is a better transfection reagent than calcium phosphate for the production of recombinant AAV in both HEK293 and HeLa cells. In addition, when both HeLa and XDC293 cells were by our method, HeLa cells in the absence of E1A generated three-fold more recombinant AAV than XDC293 cells, which constitutively express E1A.

Downstream process engineering evaluation of transgenic soybean seeds as host for recombinant protein production

The advantages of using seeds for the production of recombinant proteins with plant-based expression system has been demonstrated by several researchers. The high productivity makes soybean a potential system for large-scale recombinant protein production. However, there is a lack of detailed engineering studies of the downstream process (DSP) of recombinant proteins produced in transgenic soybean. In this work, we evaluated the use of transgenic soybean seeds as hosts for the production of recombinant proteins from a downstream process (DSP) engineering standpoint. Recombinant β-glucuronidase (rGUS), was used as a model for extraction and purification studies. This study showed, that even a protein with acidic p I (rGUS) can be successfully separated from native soybean proteins, which also have acidic p I. Maximum GUS specific activity (9.5 × 10 3 U/mg) with high total activity recovery (8.9 × 10 4 U/mL) was obtained using a simple extraction solution composed of 50 mmol/L citrate buffer at pH 5.25. Purification of rGUS was evaluated by a two-step chromatographic procedure – anion-exchange followed by hydrophobic interaction chromatography – which was compared to the purification of rGUS from transgenic corn and canola. Overall purification factor and activity recovery obtained were 97.3 and 110% (a value higher than 100% probably due to removal of an inhibitor). Comparison of this study with similar ones made with corn and canola seeds indicates that in terms of DSP soybean seeds can be considered a potentially viable plant system for the production of recombinant proteins.

Cloning, Characterization and Tissue Specific Expression of Amur Tiger (Panthera tigris altaica) IGF-I

Insulin-like growth factor I (IGF-I) plays an important role in regulating gonad function, which is essential for normal reproduction in animals, especially in sexual receptivity and reproductive behavior. In this study, a cDNA encoding Amur tiger (Panthera tigris altaica) IGF-I was isolated from liver total RNA using RT-PCR. The IGF-I cDNA of Amur tiger (ATIGF-I) was highly homologous to that of other animals, 84.8% to rat, 93.7% to human and horse. Alignment analysis showed that the cysteine residues and many amino acid residues of putative mature ATIGF-I are highly conserved in mammalian species, confirming the high sequence homology observed in other species. DNA encoding the mature ATIGF-I peptide was ligated with pET-DsbA expression vector and highly expressed in Escherichia coli BL21 with IPTG induction. The recombinant proteins expressed existed mostly in the soluble protein fraction, and were purified with metal affinity resins. Western blotting confirmed that the recombinant proteins reacted with antibodies against IGF-I. The results obtained here should be useful for large-scale production of biological active ATIGF-I protein, as well as for further research on growth, development, and reproduction in the Amur tiger. Tissue specific expression of ATIGF-I mRNA in the Amur tiger was examined by reverse transcription-polymerase chain reaction (RT-PCR), The major ATIGF-I mRNA expression tissue was the liver, while medium signals were found in the uterus, ovary, and pituitary, and minor signals were detected in various tissues including the heart, spleen, pancreas, and kidney. The results indicate that IGF-I might play an important role in the reproductive system and in cub development in the Amur tiger.

A KDEL‐tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds

Transgenic plants are attractive biological systems for the large-scale production of pharmaceutical proteins. In particular, seeds offer special advantages, such as ease of handling and long-term stable storage. Nevertheless, most of the studies of the expression of antibodies in plants have been performed in leaves. We report the expression of a secreted (sec-Ab) or KDEL-tagged (Ab-KDEL) mutant of the 14D9 monoclonal antibody in transgenic tobacco leaves and seeds. Although the KDEL sequence has little effect on the accumulation of the antibody in leaves, it leads to a higher antibody yield in seeds. sec-Ab(Leaf) purified from leaf contains complex N-glycans, including Lewis(a) epitopes, as typically found in extracellular glycoproteins. In contrast, Ab-KDEL(Leaf) bears only high-mannose-type oligosaccharides (mostly Man 7 and 8) consistent with an efficient endoplasmic reticulum (ER) retention/cis-Golgi retrieval of the antibody. sec-Ab and Ab-KDEL gamma chains purified from seeds are cleaved by proteases and contain complex N-glycans indicating maturation in the late Golgi compartments. Consistent with glycosylation of the protein, Ab-KDEL(Seed) was partially secreted and sorted to protein storage vacuoles (PSVs) in seeds and not found in the ER. This dual targeting may be due to KDEL-mediated targeting to the PSV and to a partial saturation of the vacuolar sorting machinery. Taken together, our results reveal important differences in the ER retention and vacuolar sorting machinery between leaves and seeds. In addition, we demonstrate that a plant-made antibody with triantennary high-mannose-type N-glycans has similar Fab functionality to its counterpart with biantennary complex N-glycans, but the former antibody interacts with protein A in a stronger manner and is more immunogenic than the latter. Such differences could be related to a variable immunoglobulin G (IgG)-Fc folding that would depend on the size of the N-glycan.

Refolding, purification and characterization of replication-initiator protein from soybean-infecting geminivirus

The replication-initiator protein (Rep) from a soybean-infecting geminivirus was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). In spite of the presence of the highly soluble MBP as the fusion partner, the overexpressed MBP-Rep fusion protein formed insoluble inclusion bodies. The protein was solubilized from the inclusion bodies and refolded. The refolded MBP-Rep protein was purified using ion exchange and amylose affinity chromatography. The activity of the purified MBP-Rep was assessed using an in vitro cleavage assay. Soluble and stable MBP-Rep protein was obtained in high abundance, providing the feasibility of large-scale production of active Rep protein for functional characterization and X-ray crystallographic structure determination.

Production of a Cellulase in Silkworm Larvae using a Recombinant Bombyx mori nucleopolyhedrovirus lacking the Virus-encoded Chitinase and Cathepsin Genes

The expression efficiency of an insect-derived cellulase was assayed in silkworm larvae infected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) mutants lacking the virus-encoded chitinase (chiA) and/or cathepsin (v-cath) genes. Expression was increased by approx. 10% in mutants lacking chiA or v-cath and 17% in a mutant lacking both chiA and v-cath compared with that of the unmodified recombinant BmNPV. The recombinant BmNPV lacking both chiA and v-cath can therefore be used for a large-scale production of foreign proteins in silkworms.