Baculovirus expression system: An alternative for producing catalytically active human PTP-1B

Protein tyrosine phosphatases (PTPs) play multiple roles in many physiological processes. Over-expression of the PTPs has been shown to be associated with cellular toxicity, which may also lead to the deletion of the respective gene from stable cell clones. We also observed that PTP-1B over-expression in CHO and HEK293 stable cell clones led to cytotoxicity and low revival rates during clone generation and maintenance. To address these issues, bacmid transposition technology was utilized to generate recombinant PTP-1B baculovirus, and Spodoptera frugiperda (Sf9 and Sf21) insect cell lines were infected with the virus. The data obtained on expression and activity of the PTP-1B highlights clear advantage of the recombinant baculovi-rus-insect cell expression system over the mammalian cell line technique due to increase in enzyme activity, strongly inhibited by phosphatase specific inhibitor RK682. Possible application of the expression system for producing active enzymes in bulk quantity for a new drug discovery is also discussed.     

Constitutive expression of functionally active protease-activated receptors 1 and 2 in human conjunctival epithelial cells

Protease-activated receptors (PARs) are G-protein-coupled receptors which initiate inflammatory responses when activated by specific serine proteases. This study was conducted to examine whether human conjunctival epithelial cells (HCECs) express functionally active PAR1 and PAR2 using Chang conjunctival epithelial cells as in vitro model. We performed RT-PCR and immunofluorescence analyses to determine the expression of PAR1 and PAR2, and monitored the production of IL-6 after activating HCECs with PAR1 activating agents (thrombin or TFLLRN) or PAR2 activating agents (tryptase, trypsin, or SLIGKV). The results show that HCECs constitutively express PAR1 and PAR2 mRNA and proteins, and produce significant amounts of IL-6 when incubated with specific PAR-activating enzymes or agonist peptides. Thrombin- and tryptase-induced HCEC activation was blocked by PAR1 and PAR2 neutralizing antibodies, respectively, and by specific enzyme inhibitors. The constitutive expression of PAR1 and PAR2, and their activation by thrombin and tryptase, respectively, may have important implications in ocular inflammation.     

High level expression of functionally active human lactoferrin in silkworm larvae

In this paper, recombinant human lactoferrin (rhLf) was expressed very well using Bombyx mori nuclear polyhedrosis baculovirus expression system. Infection of silkworm larvae with recombinant virus, vBm-hLf, the rhLf was efficiently secreted into larvae hemolymph and the concentration of product purified was about 65 microg/ml. The isolated rhLf molecular mass was approximately 78 kDa, lower than that of the human lactoferrin (hLf) standards, which may be due to incomplete glycosylation or protein degradation. Furthermore, the rhLf was characterized and its biological activities were evaluated by in vivo bioassay using dextran sodium sulfate (DSS)-induced colitis mouse model that mimics some characteristics of colitis disease in human. We conclude that silkworm expression system can be used successfully to express functional human lactoferrin.     

An inducible expression system for the production of human lactoferrin in Aspergillus nidulans.

The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described. The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (alcA) promoter. Recombinant hLF (re-hLF) is produced at levels up to 5 micrograms/ml. Approximately 30% of the re-hLF produced in this system is secreted into the growth medium. The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity. Furthermore, re-hLF is functional by the criterion of iron-binding capacity. The A. nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.     

An efficient system for small protein expression and refolding

The low expression yield and poor refolding efficiency of small recombinant proteins expressed in Escherichia coli have continued to hinder the large-scale purification of such proteins for structural and biological investigations. A system based on a small fusion partner, the B1 domain of Streptococcal protein G (GB1), was utilized to overcome this problem. We have tested this system on a small cysteine-rich toxin, mutant myotoxin alpha (MyoP20G). The highly expressed fusion protein was refolded using an unfolding/refolding protocol. Due to the small size of GB1, we were able to monitor the unfolding/refolding status by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The final product yielded well-resolved NMR spectra, with a topology corresponding to the natural product. We conclude that GB1 not only increases the expression level but also enhances the refolding of small proteins.     

Establishment of a microcarrier culture system with serial sub-cultivation for functionally active human endothelial cells

A microcarrier culture system was established for a large-scale production of functional human endothelial cells. It has been difficult to cultivate human endothelial cells in large quantities for the reasons that specific growth factor and extracellular matrix are required for the survival and proliferation of the cells and the life span of the primary cells are limited. A lot of studies have reported that the shear stress gives significant influences on the structure, growth rate and biological functions of endothelial cells. We aimed to develop a convenient microcarrier culture system for human endothelial cells which can reproduce the flow effects experienced in vivo or in vitro. In 200 mL volume culture, human umbilical vein endothelial cells (HUVEC) could be serially sub-cultivated by optimizing the culture conditions such as shear strength, growth factor, beads and seeding cell concentration, serum concentration, and passage timing. The growth rate was enhanced depending on the shear strength and the life span of the cells was elongated until over 43PDL which is much longer than those of monolayer cultures. The cells maintained the diploidy of over 80% without obvious abnormal changes in the chromosomes. The serially sub-cultured microcarrier cells maintained various endothelial cell functions such as the syntheses of von Willebrand factor (vWf), prostacyclin and other biological substances, the expression of CD31, and the VEGF(165) dependent growth characteristic. The synthesis of biological products was affected by shear strength. In the case of prostacyclin, a different synthesis response was observed between steady flow and transiently reduced shear strength. The synthesis of endothelin-1 (ET-1) was down-regulated by increase of shear strength different from those of other products. The culture system was scaled up until 2 L volume under the optimum DO control. The cells synthesized IL-6 in response to shear strength. These results indicate that the established microcarrier system might be able to contribute to the supply of functional human endothelial cells for various medical applications such as the reconstruction of injured blood vessels caused by atherosclerosis or restenosis of coronary arteries after angioplasty, and the construction of an anti-coagulable artificial blood vessel or an artificial skin with good transplant-ability.     

Preparation of functionally active recombinant human interleukin-6

The gene of human interleukin-6 (hIL-6) with an additional 20 amino acids on the N-end, including six histidine residues, was cloned into the expression plasmid pET28b(+). The conditions were elaborated for preparing highly active protein both using denaturing agents and without them. Application of a dialysis cascade allowed us to prepare a functionally active hIL-6 of 90-95% purity with the yield of 3 mg from liter of the cell culture. The highest activity was detected by ELISA in the preparation obtained without denaturing agents. The functional activity of hIL-6 was studied by flow cytofluorimetry. Addition of hIL-6 to the cells of immortal lines of human multiple myeloma resulted in dimerization of the gp130 receptor molecule.     

An expression system for the efficient incorporation of an expanded set of tryptophan analogues

Biosynthetic incorporation of tryptophan (Trp) analogues in recombinant proteins using an E. coli Trp auxotroph expression host is limited to analogues modified with a small substituent like a fluoro atom or a hydroxyl or amine group. We report here the efficient incorporation (>89 %) of chloro- and bromo atoms containing Trp analogues in alloproteins at high expression levels using a Lactococcus lactis Trp auxotroph strain. This result was only obtained after coexpression of the enzyme tryptophanyl-tRNA synthetase (TrpRS) of L. lactis, an enzyme believed to show a more relaxed substrate specificity than TrpRS from E. coli. Chloro- and bromo-Trps are attractive intrinsic phosphorescence probes as these Trp analogues are much less sensitive for quenchers in the medium, like oxygen, than Trp. Coexpression of TrpRS was also essential for the biosynthetic incorporation (94 %) of the Trp analogue 5,6 difluoroTrp. This makes our expression system ideally suited to generate a set of methyl- and fluoro-substituted Trp analogue-containing alloproteins in high yield for investigating the involvement of the Trp residue in cation-pi or pi–pi interactions. Taken together, the presented Trp auxotroph expression system features the most relaxed specificity for Trp analogue structures reported to date and gives a high alloprotein yield.     

Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system

We have characterized the promoter region of the human elongation factor 1 alpha-encoding gene (EF-1 alpha) and developed a versatile expression system which has a wide host range and a high efficiency of gene expression. To identify the promoter region of the EF-1 alpha gene necessary for efficient gene expression, we constructed four pEF-CAT plasmids that have the bacterial cat gene fused to four different sites of the human EF-1 alpha gene: (i) ligated to the end of the TATA box (pEF220-CAT); (ii) ligated in exon 1 (pEF204-CAT and pEF233-CAT), and (iii) ligated in exon 2 (pEF321-CAT). All the pEF-CAT plasmids were highly expressed in all the cell types tested, including fibroblasts and lymphoid cells. Plasmid pEF321-CAT, which contains the first exon and the first intron, gave the highest level of cat expression. Plasmids pEF204- and pEF233-CAT, which contain part of the first exon but do not contain the first intron, were less efficient in cat expression than was pEF321-CAT. Plasmid pEF220-CAT, which lacks both the first exon and the first intron, was the least efficient. Plasmid pEF321-CAT was several- to 100-fold more efficient in cat expression than plasmid pSV2-CAT depending on the recipient cell types. The promoter of pEF321 plasmid also directed the stable expression of the bacterial neo gene more efficiently than the promoter of the simian virus 40 (SV40) early gene or the long terminal repeat of Rous sarcoma virus. Using this system, the SV40 early gene and the cDNA encoding human CD4 were also expressed efficiently.     

Highly purified, functionally active human fibronectin preparation

Fibronectin has been purified by gelatin-Sepharose affinity chromatography from fresh frozen human plasma. The bound fibronectin was eluted with 3 M urea. The purity of the fibronectin obtained has been checked on (immunoelectrophoresis, polyacrylamide gel electrophoresis, FPLC). Biological activity of the purified molecule has been monitored by means of three assays: quantitation of the gelatin-binding activity by ELISA, quantitation of the fibronectin-mediated attachment of fibroblasts on plastic and evaluation of the opsonic activity (uptake of gelatin latex particles by a murine macrophage line). When deep-frozen, fibronectin retains all of its properties. This highly purified and functional fibronectin fulfills the basic requirements for a standard reagent. It will allow to investigate physicochemical and functional alterations of various fibronectins.     

Constitutively active PDX1 induced efficient insulin production in adult murine liver

To generate insulin-producing cells in the liver, recombinant adenovirus containing a constitutively active mutant of PDX1 (PDX1-VP16), designed to activate target genes without the need for protein partners, was prepared and administered intravenously to streptozotocin (STZ)-treated diabetic mice. The effects were compared with those of administering wild-type PDX1 (wt-PDX1) adenovirus. Administration of these adenoviruses at 2x10(8)pfu induced similar levels of PDX1 protein expression in the liver. While wt-PDX1 expression exerted small effects on blood glucose levels, treatment with PDX1-VP16 adenovirus efficiently induced insulin production in hepatocytes, resulting in reversal of STZ-induced hyperglycemia. The effects were sustained through day 40 when exogenous PDX1-VP16 protein expression was undetectable in the liver. Endogenous PDX1 protein came to be expressed in the liver, which is likely to be the mechanism underlying the sustained effects. On the other hand, albumin and transferrin expressions were observed in insulin-producing cells in the liver, suggesting preservation of hepatocytic functions. Thus, transient expression of an active mutant of PDX1 in the liver induced sustained PDX1 and insulin expressions without loss of hepatocytic function.     

An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha

An efficient process was developed for the low-cost production of phytases using Hansenula polymorpha. Glucose or glucose syrups, previously reported as repressive substrates, were used as main carbon sources during fermentation. Glucose was even the most productive substrate for high-level production of phytases. Compared with the process using glycerol, the standard carbon source used for this process until now, the use of glucose led to a reduction of more than 80% in the raw materials costs. In addition, exceptionally high concentrations of active enzyme (up to 13.5 g/L) were obtained in the medium, with phytase representing over 97% of the total accumulated protein. These levels greatly exceed those reported so far for any yeast-based expression system. Very efficient downstream processing procedures were developed with product recovery yields over 90%. Both the fermentation and downstream processing were successfully tested in pilot scale up to 2000 L. As a result, H. polymorpha can be used as a highly competitive system for low-cost phytase production.     

Expression of functionally active sialylated human erythropoietin in plants

Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO. cDNA constructs were delivered to Nicotiana benthamiana (N. benthamiana) and transiently expressed by a viral based expression system. Expression levels up to 85 mg rhEPO/kg fresh leaf material were achieved. Moreover, co-expression of rhEPO with six mammalian genes required for in planta protein sialylation resulted in the synthesis of rhEPO decorated mainly with bisialylated N-glycans (NaNa), the most abundant glycoform of circulating hEPO in patients with anemia. A newly established peptide tag (ELDKWA) fused to hEPO was particularly well-suited for purification of the recombinant hormone based on immunoaffinity. Subsequent lectin chromatography allowed enrichment of exclusively sialylated rhEPO. All plant-derived glycoforms exhibited high biological activity as determined by a cell-based receptor-binding assay. The generation of rhEPO carrying largely homogeneous glycosylation profiles (GnGnXF, GnGn, and NaNa) will facilitate further investigation of functionalities with potential implications for medical applications.     

An efficient system for high-level expression and easy purification of authentic recombinant proteins

Expression of recombinant proteins as fusions to the eukaryotic protein ubiquitin has been found to significantly increase the yield of unstable or poorly expressed proteins. The benefit of this technique is further enhanced by the availability of naturally occurring deubiquitylating enzymes, which remove ubiquitin from the fusion product. However, the versatility of the system has been constrained due to the lack of a robust, easily purified deubiquitylating enzyme. Here we report the development of an efficient expression system, utilizing the ubiquitin fusion technique, which allows convenient high yield and easy purification of authentic protein. An Escherichia coli vector (pHUE) was constructed for the expression of proteins as histidine-tagged ubiquitin fusions, and a histidine-tagged deubiquitylating enzyme to cleave these fusions was expressed and purified. The expression system was tested using several proteins varying in size and complexity. These results indicate that this procedure will be suitable for the expression and rapid purification of a broad range of proteins and peptides, and should be amenable to high-throughput applications.     

A mammalian expression system for rapid production and purification of active MAP kinase phosphatases

Expression of enzymatically active mammalian proteins in Escherichia coli can proven to be a challenging task due to poor solubility, improper folding, and lack of adequate posttranslational modification. Expression of mammalian proteins using baculovirus or yeast systems is time-consuming and may also be subject to inadequate modification. In order to overcome these technical difficulties, we have developed a mammalian expression system for the convenient subcloning of cDNA fragments, high-level expression, and one-step purification of enzymatically active proteins. The mammalian expression vector pEBG that expresses glutathione S-transferase fusion proteins was modified to create an SrfI restriction site in the multiple cloning site. The protein coding sequences of MAP kinase phosphatase-1 (MKP-1), MAP kinase phosphatase-2 (MKP-2), and the tumor suppressor PTEN were PCR-amplified using Pfu DNA polymerase and cloned into the SrfI site through SrfI digestion-coupled ligation. The resulting plasmids were transiently transfected into 293T cells using FuGENE 6 transfection reagent. Forty eight hours after transfection, cells were harvested and bioactive recombinant proteins were purified by glutathione-Sepharose beads. Protein yield, which ranged from 200 to 700 microg, was more than adequate for biochemical studies. The usefulness of this versatile system for studying protein function and its potential application for proteomics research are discussed.     

A new efficient expression system for Bacillus and its application to production of recombinant phytase

A new expression system was developed for Bacillus subtilis.This system uses a shuttle vector (B. subtilis– Eschericia coli) carrying a phosphate starvation-inducible promoter (pst) and on a fed-batch cultivation strategy. The pst-promoter proved to be very strong and retain its tight regulation also when present on a multi-copy plasmid. The expression system developed showed promising results when applied to the production of recombinant Bacillusphytase – phytase activity at the end of cultivation reached 28.7 U ml^–1.     

An efficient heat-inducible Bacillus subtilis bacteriophage 105 expression and secretion system for the production of the Streptomyces clavuligerus beta-lactamase inhibitory protein (BLIP)

The Streptomyces clavuligerus beta-lactamase inhibitory protein (BLIP) has been shown to be a potent inhibitor of class A beta-lactamases including the Escherichia coli TEM-1 beta-lactamase (Ki = 0.6 nM). A heat-inducible BLIP expression system was constructed based on a derivative of Bacillus subtilis phage phi105. The recombinant BLIP produced by this system was secreted to the culture medium, purified to homogeneity, and fully active. We have shown that the signal peptide of BLIP functions well in B. subtilis to secrete BLIP out of the cells, which facilitates purification. The absence of a His-tag also avoids the activity and structure of BLIP being altered. An unprecedented high yield of recoverable protein in culture supernatant (3.6mg of >95% pure BLIP/l culture) was achieved by a simple purification protocol. We have developed an efficient production process in which the culture time before heat-induction was 3-4h and the culture supernatant could be collected 5h after induction. This total time of 8-9h is considered to be very short compared to that of the native S. clavuligerus culturing (60-70h). We achieved a very efficient BLIP production rate of 0.8-0.9mg/l/h. Heterologous gene expression was tightly controlled and no production of BLIP was observed before heat-induction, suggesting that cell density can be further increased to improve enzyme yield.