Expression of kringle 5 domain of human plasminogen in Pichia pastoris

The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelia cell specifically, and shows promise in antiangiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When expressed in E. coli, recombinant, kringle 5 deposited mainly as inactive, insoluble inclusion bodies and the refolding yield was also low. In the present study, human kringle 5 encoding gene was cloned into secretory plasmid pPIC9K and then integrated into Pichia pastoris genome for expression. On methanol induction, biologically active recombinant kringle 5 was expressed and secreted into the culture medium by the integrated Pichia pastoris with the expression level around 30mg/L of yeast culture. After a simple and economical three-step purification protocol, namely precipitation, DEAE ion exchange chromatography, and gel filtration, the recombinant kringle 5 was purified to homogeneity, with the yield of 7.5 mg/liter yeast culture.     

Purification and biological characterization of bacterially expressed recombinant buffalo prolactin

Recombinant prolactin (PRL) from water buffalo (Bubalus bubalis) has been cloned and expressed in a prokaryotic expression system. The hormone was also successfully refolded into a biologically active form. Total RNA was purified from buffalo pituitaries and the buPRL cDNA was synthesized using primers designed on bovine PRL sequence. This prolactin cDNA was cloned in a pET 28a vector and expressed in Escherichia coli strain BL21(DE3)pLysS. Most of the expressed protein was present as insoluble inclusion bodies. The inclusion bodies were solubilized and buPRL was purified by Ni-NTA column. The purified protein was refolded by gradually decreasing the concentration of denaturant during dialysis. Total yield of the refolded and soluble prolactin was 22?mg/L from 100?mL bacterial culture in LB medium. The recombinant prolactin was as active as native prolactin in stimulating growth of Nb2 lymphoma cells.     

Characterization of Recombinant <Emphasis Type="Italic">Thermococcus kodakaraensis</Emphasis> (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.     

Cloning and expression of recombinant macrophage-colony stimulating factor - A progressive strategy for economical production

Macrophage-colony stimulating factor (M-CSF) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, recombinant M-CSF (rM-CSF) became widely used as a biological research reagent in bone marrow stimulations, vaccine development, gene therapy approaches, and stem cell mobilization. rM-CSF is a glycoprotein that activates and enhances the differentiation and survival of macrophages, which play a key role in the osteoclastogenetic response. Here, we describe the construction of the gene encoding rM-CSF, its cloning, and expression in Escherichia coli, as well as the purification of rM-CSF protein, and its activity in a biological assay in mouse bone marrow cells. Our results show that the combination of experimental strategies employed to obtain recombinant rM-CSF can yield a biologically active protein, and may be useful when scaling-up production of other biologically similar proteins.     

Enhanced soluble production of biologically active recombinant human p38 mitogen-activated-protein kinase (MAPK) in Escherichia coli

The conditions were optimized for maximum soluble yield of biologically active recombinant p38alpha mitogen activated protein kinase (MAPK) vis-à-vis insoluble fraction (inclusion body formation). This study reports a rapid, economical and single step purification process for the overproduction of GST tagged p38alpha MAPK. A yield of 18 mg of highly purified and soluble protein per liter of bacterial culture within 6 h timeframe was achieved. The purified protein was found to be biologically suitable for phosphorylation by upstream kinases and was catalytically active. We further demonstrated that our in-house p38alpha MAPK is more potent (>30%) than a commercially available enzyme.     

Production of a recombinant human basic fibroblast growth factor with a collagen binding domain

Summary Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney, and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-Fl) fromEscherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of a purification tag, a protease-sensitive linker and collagen binding domain, and a cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains the collagen binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured by a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a nickel-nitrilotriacetic acid metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of human vein endothelial cells, monitored by [³H]thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. The high-affinity binding was demonstrated by the binding of [³H]collagen to the rhbFGF-F2 protein immobilized on a Ni-nitrilotriacetic acid column. The rhbFGF-F2 fusion protein bound to collagen-coated surfaces with high affinity. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for their high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type 1 collagen. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins for specific biomedical applications.     

Production of an active Mus musculus IL-3 using updated silkworm-based baculovirus expression vector system

Due to the biological significance and therapeutic potential of Interleukin-3 (IL-3) secreted mainly by activated T cells, various protein expression systems have been challenged to produce recombinant IL3 to meet the increasing demands worldwide. Recently, we established an updated silkworm-based baculovirus expression vector system (silkworm-BEVS), which in most cases, produces eukaryotic proteins in biological or enzymatical active forms with considerable amounts. We attempted to reconstruct and express a recombinant mouse IL-3 (rMmIL-3) with C-terminal His8-Strep tags in silkworm-BEVS in the current study. From our results, we gained an active glycosylated rMmIL-3 protein in a substantial amount and quality. As compared with the E. coli expression system, silkworm-BEVS is a better choice regarding the glycosylations attached in rMmIL-3 and up-scalable system in case that a commercial amount is required in the future. Collectively, our method shares an excellent model to produce interleukin molecular for approaching pharmaceutical applications.     

Secretory production of hGM-CSF with a high specific biological activity by transgenic plant cell suspension culture

The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically active hGM-CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about 180 μg/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to 270 μg/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than that of a commercially available preparation fromE. coli.     

High-level expression, purification, and characterization of non-tagged Aspergillus flavus urate oxidase in Escherichia coli

The entire encoding region for Aspergillus flavus uricase was cloned into pET-32a and expressed in Escherichia coli BL21 (DE3). The uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form. A scalable process aimed to produce and purify multi-gram quantities of highly pure, recombinant urate oxidase (rUox) from E. coli was developed. The rUox protein was produced in a 30 L fermentor containing 25 L of 2x YT medium and purified to >99% purity using hydrophobic interaction, anion-exchange, and gel filtered chromatography. The final yield of purified rUox from fermentation resulted in approximately 27 g of highly pure, biologically active rUox per kg of cell paste (approximately 238 mg/8.8 g cell paste/L). The results presented here exhibit the ability to generate multi-gram quantities of rUox from E. coli that may be used for the development of pharmaceutics of reducing the hyperuricemia.     

High-level expression and purification of human xylosyltransferase I in High Five insect cells as biochemically active form

Human xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to consensus serine residues of proteoglycan core proteins. Expression of a soluble form of recombinant histidine-tagged XT-I (rXT-I-HIS) was accomplished at a high level with High Five/pCG255-1 insect cells in suspension culture. The recombinant protein was purified to homogeneity by a combination of heparin affinity chromatography and metal (Ni(2+)) chelate affinity chromatography. Using the modern technique of perfusion chromatography, a rapid procedure for purification of the rXT-I-HIS from insect cell culture supernatant was developed. The purified, biologically active enzyme was homogeneous on SDS-PAGE, was detected with anti-XT-I-antibodies, and had the expected tryptic fragment mass spectrum. N-terminal amino acid sequencing demonstrated that the N-terminal signal sequence of the expressed protein was quantitatively cleaved. The total yield of the enzyme after purification was 18% and resulted in a specific XT-I activity of 7.9mU/mg. The K(m) of the enzyme for recombinant [Val(36),Val(38)](delta1),[Gly(92),Ile(94)](delta2)bikunin was 0.8microM. About 5mg purified enzyme could be obtained from 1L cell culture supernatant. The availability of substantial quantities of active, homogeneous enzyme will be of help in future biochemical and biophysical characterization of XT-I and for the development of a immunological XT-I assay.     

High levels of stable phytase accumulate in the culture medium of transgenic Medicago truncatula cell suspension cultures

The use of plants for production of recombinant proteins is becoming widely accepted. More recently, plant cell cultures have been proposed as valuable systems for producing a wide range of biologically active proteins. Such systems provide certain advantages over whole plants, but yields are still considered a limitation. In this study we established a Medicago truncatula cell suspension line expressing phytase from Aspergillus niger. Phytase is an N-glycosylated enzyme that breaks down indigestible phytate, resulting in an increased availability of phosphorus and other minerals in monogastric animals and reduced levels of phosphorus output in their manure. Various production systems have previously been used to express heterologous phytase, including several plant species. In this work, remarkable amounts of enzymatically active recombinant phytase were produced and secreted into the culture medium. Recombinant phytase accumulated to at least 25 mg/L and remained stable along the growth curve, and an enriched fraction with high enzymatic activity was easily obtained. We therefore propose M. truncatula cell suspension cultures as a potential system for the production of recombinant proteins. Most importantly, we have shown that, contrary to general belief, it is possible to achieve high levels of a functional recombinant protein in plant cell culture systems.     

Production of Hepatopoietin Cn in High-Cell-Density Cultures of Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> and Detection of its Antioxygen Activity

Hepatopoietin Cn (HPPCn), a member of the leucine-rich acidic nuclear protein (LANP) family, is a novel human hepatic growth factor that stimulates DNA synthesis and hepatocyte proliferation in vivo and in vitro by activating several signaling pathways. We performed pilot-scale production of recombinant human HPPCn (rhHPPCn) protein to elucidate its role in liver protection. An Escherichia coli BL21 (DE3) strain containing a pET-24a-HPPCn expression vector was cultivated in a 40-l bioreactor. High-cell-density fed-batch fermentation using this strain yielded a high concentration of rhHPPCn protein in the form of inclusion bodies. After washing and solubilization of the protein, rhHPPCn refolding was achieved by dilution of the denaturant. Then, the renatured protein was purified chromatographically. Assessment of the biological activity of the obtained protein showed that rhHPPCn could attenuate the oxidative stress in ethanol-induced hepatocyte injury. We successfully achieved large-scale production of biologically active rhHPPCn protein and obtained yields greater than 40 mg rhHPPCn per gram dry cell weight by applying the above-mentioned renaturation and purification procedures. The obtained volumetric yield corresponds to 640 mg of biologically active rhHPPCn per liter of culture broth. Additionally, the recombinant HPPCn protein was confirmed to protect hepatocytes against ethanol-induced oxidative stress.     

Production of human mutant biologically active hepatocyte growth factor in Chinese hamster ovary cells

Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12?mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins.     

Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp

A distinct cysteine proteinase (NsCys) of northern shrimp Pandalus borealis belonging to cathepsin L subgroup of the papain superfamily has been overexpressed as a precursor form (proNsCys) in Pichia pastoris. We adopted a simple and quick procedure to generate an expression cassette by constructing a donor vector harboring proNsCys followed by recombination with an acceptor vector in a way so that the proNsCys gene was placed downstream of the methanol-inducible AOX1 promoter and alpha-mating factor signal sequence gene. In addition, we used glycerol complex medium that supported high growth of yeast before induction while induction was carried out in minimal methanol medium thereby facilitating the secreted protein to be purified with a single size-exclusion chromatography. The recombinant enzyme was purified in two enzymatically active fractions: both corresponding to mature NsCys with, however, the major one comprising two molecular species of NsCys which had their severed prodomain non-covalently attached. The overall yield was about 100 mg of crude or 60 mg of purified recombinant enzyme comprising both mature and prodomain-attached forms of NsCys per liter of yeast culture. The recombinant NsCys was biologically active as observed by gelatin zymography and its ability to cleave Z-Phe-Arg-MCA, a synthetic substrate for cathepsin L. The development of the system reported here provides a cost-effective and easy to manipulate expression system to obtain large quantities of fully functional shrimp enzyme that will enable the functional characterization of this unique enzyme for both research and industrial purposes.     

Production of human recombinant beta-interferon in the avian cell culture

The avian recombinant adenovirus of serotype 1 (CELO) was obtained. The recombinant adenovirus of serotype 1 (CELO) induces expression of human beta-interferon (IB). The expression cassette containing IB gene was placed at the right end of the CELO genome under control of hybrid promoter hEF-1alpha/HTLV. The resulting recombinant adenovirus CELO-IB transduced the avian cell culture LMH. The level of production of the recombinant IB was 0.15 micro/ml. The IB protein yield after affine chromatography purification using Ni-NTA agarose was 50%. The biological activity of the purified IB was high (7.8 x 10(8) MU/microg protein). The purified IB inhibited replication of murine encephalomyocarditis virus (VMEC) in cell culture of human diploid fibroblasts (HDF). Thus, expression system based on avian cell culture is an effective system for producing biologically active protein of human interferon beta.     

Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris

Antifreeze proteins and antifreeze glycoproteins are structurally diverse molecules that share a common property in binding to ice crystals and inhibiting ice crystal growth. Type II fish antifreeze protein of Atlantic herring (Clupea harengus harengus) is unique in its requirement of Ca(2+) for antifreeze activity. In this study, we utilized the secretion vector pGAPZalpha A to express recombinant herring antifreeze protein (WT) and a fusion protein with a C-terminal six-histidine tag (WT-6H) in yeast Pichia pastoris wild-type strain X-33 or protease-deficient strain SMD1168H. Both recombinant proteins were secreted into the culture medium and properly folded and functioned as the native herring antifreeze protein. Furthermore, our studies demonstrated that expression at a lower temperature increased the yield of the recombinant protein dramatically, which might be due to the enhanced protein folding pathway, as well as increased cell viability at lower temperature. These data suggested that P. pastoris is a useful system for the production of soluble and biologically active herring antifreeze protein required for structural and functional studies.     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

Expression of human VEGF165 in silkworm (Bombyx mori L.) by using a recombinant baculovirus and its bioactivity assay

Silkworm larva has a lot of advantages as a "biofactory" to produce recombinant protein. A recombinant baculovirus, carrying cDNA encoding the 165 amino-acid long isoform of human vascular endothelial growth factor (VEGF) was successfully constructed for the large-scale production of this protein using silkworms as an in vivo host. The fifth-instar silkworm larvae were inoculated with the recombinant virus. Time-course expression analysis indicated that the expression level was highest at around 80 h post-infection and the recombinant protein was found mainly in the haemolymph. Therefore, the hemolymph was collected from the infected larvae and the recombinant protein was purified by using Nickel affinity chromatography under native condition. The expression level was estimated to be as high as approximately 426 microg per larva. Furthermore, the recombinant protein was characterized and was found biologically active in inducing endothelial cell proliferation in vitro.