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PubMed1998

Predictable deuteration of recombinant proteins expressed in Escherichia coli.

Random deuteration of recombinant proteins in Escherichia coli is widely used for protein structure determination by nuclear magnetic resonance (NMR). It is desirable to predict accurately the degree of deuteration because each NMR experiment benefits from a different level of deuteration. The method described here which uses [2H]2O and glucose as the sole carbon and deuterium sources is an alternative for a previously published procedure using acetate and [2H]2O (Venter et al., J. Biomol. NMR 5, 339-344, 1995) and it is of advantage for proteins that do not express well using acetate. While the deuteration degree with acetate is approximately linear with the [2H]2O content in the medium, the use of glucose leads to deviations up to 19%, which is analyzed systematically here. With [2H]2O as the sole deuterium source 0-86% of the chemically nonexchangeable hydrogen atoms can be deuterated. Higher levels of deuteration require perdeuterated glucose in combination with [2H]2O. As an example, recombinant peptide deformylase from Bacillus subtilis was overexpressed, deuterated to various degrees, purified, and analyzed by mass spectrometry and NMR.

Amidohydrolases↗

PubMed1985

Expression of recombinant vaccinia virus-derived alphavirus proteins in mosquito cells.

A recombinant vaccinia virus strain which contains and expresses a 26S cDNA insert encoding Sindbis virus structural proteins (VV:3S) was used to infect a continuous line of Aedes albopictus mosquito cells. There were not visible cytopathic effects due to the virus infection and the cells continued to grow normally. However, examination of the proteins present in the cytoplasm of the infected cells with Sindbis virus-specific antisera revealed that Sindbis virus proteins were being synthesized and processed. These results are discussed with respect to vaccinia virus as a non-lethal expression vector to deliver and express eukaryotic genetic information in insect cell systems and using this system (VV:3S) to dissect various facets of togavirus-insect cell interactions.

Aedes↗

PubMed1994

pJC20 and pJC40--two high-copy-number vectors for T7 RNA polymerase-dependent expression of recombinant genes in Escherichia coli.

We report the construction of two plasmid vectors, pJC20 and pJC40, for the expression of recombinant genes in Escherichia coli under the control of T7 RNA polymerase. Their small sizes of ca. 2.4 kb ease the subcloning of large inserts and the high copy numbers obtained result in satisfactory yields in all plasmid preparations. A multiple-cloning site offers sites for directional cloning and nested deletions. In addition, pJC40 encodes a cleavable amino-terminal histidine tail of 10 residues which is added to the gene product, thus allowing purification by metal chelate chromatography. Observed expression yields are in the range of 10% of total bacterial protein for all genes tested in our laboratory.

Animals↗

PubMed2004

[Soluble expression of recombinant immunotoxin against human bladder carcinoma and its anti-tumor activity].

AIM: To construct soluble expression vector of the recombinant immunotoxin against human bladder carcinoma and express and purify the immunotoxin with high biological activity. METHODS: The gene fragment of the immunotoxin containing the genes encoding the humanized single-chain antibody against human bladder carcinoma and the truncated and modified form of Pseudomonas exotoxin(PE) named PE38/KDEL was digested from the plasmid pABDIT and inserted into the expression vector pTMFK containing the FkpA gene. The recombinant plasmid was used to transform the E.coli strain BL21(DE3)-Star and the immunotoxin was co-expressed with the FkpA which served as the folding assistant factor. The immunotoxin was purified through Ni-NTA agarose and anti-PEAb Sepharose 4B columns. The binding activity of the immunotoxin to the antigen on BIU-87 cells was detected by ELISA. The specific cytotoxic effect of the immunotoxin against BIU-87 cells was assessed by MTT colorimetry. RESULTS: The immunotoxin was over-expressed in soluble form in E.coli. The immunotoxin showed good binding activity to the membrane antigen on BIU-87 cells. The result of MTT assay demonstrated that purified recombinant immunotoxin could specifically kill BIU-87 cells. CONCLUSION: The immunotoxin was obtained with specific cytotoxicity to human bladder cancer cells, which lays the foundation for the further application of the immunotoxin in the target therapy of human bladder carcinoma.

ADP Ribose Transferases↗

PubMed2003

[Expression of recombinant human zona pellucida-3 protein (rhZP3) in Pichia pastoris].

Human Zona Pellucida(ZP), which is a complex matrix surrounding oocytes,is comprised of three immunologically distinct glycoproteins(hZP1, hZP2 and hZP3). Because hZP3 possesses the sperm receptor activity and the acrosome-inducing activity, it has long been used as a candidate antigen to develop an immunocontraceptive vaccine. However, a large amount of native hZP3 protein is unavailable. It is an effective way to express hZP3 protein directly in vitro. Nevertheless, it had been reported that the rhZP3 protein produced in Pichia pastoris was not secreted but accumulated in the cells and could only be purified after being solubilized by strong denaturants. More unfortunately, after purification the final product required 6mol/L urea to maintain solubility. An improved project was advanced with the aim to express secreted and soluble rhZP3 protein in yeast. In this study, the fragment of hZP3 cDNA coding for aa 23 - 408, which the N-terminal leader was removed and most of the C-terminal transmembrane-like domain was reserved, was amplified by two PCR primers including EcoR I and Not I sites respectively and a His6 codon cassette was added to 5'-terminal. The hZP3 insert was incorporated into expression vector pPIC9K. The resulting recombinant yeast expression vector was designated pPIC9K-rhZP3. Linearized pPIC9K-rhZP3 was transformed into Pichia pastoris. After G418 selection, the recombinant Pichia pastoris strains were identified by PCR and the rhZP3 was expressed following the manufacturer' s protocol. Following induction with methanol, the rhZP3 protein was secreted and dissolved into the culture supernatant. SDS-PAGE and Western blot analyses showed that the apparent molecular weight of the expressed rhPZ3 proteins in yeast was smaller and a little size heterogeneity than native ones; after purified with Ni-chelating affinity chromatography, the final product's apparent molecular weight was about 32 - 34KD and their yield more than 20mg/L. We supposed that the C-terminal transmembrane-like domain be useful for secretion of rhZP3 into the culture supernatant and the expressed rhZP3 protein be incompletely digested by proteinases of Pichia into shorter fragments which all were glycosylated inhomogeneously. Fortunately, the fragments of rhZP3 protein can be recognized in Western blot by the polyclonal antibodies to porcine ZP3 which has showed a cross-reactivity with human ZP in vitro. It will be expected that the rhZP3 protein expressed in Pichia pastoris not only has immunogencity, say, it can rise antibodies in vivo to prevent spermatozoa-ovum binding, but also does not contain ovarian factors that might be the cause of undesired side effects, e.g. ovaritis and can be used as a safe immunogen in human antifertility vaccine research.

Blotting, Western↗

PubMed1996

Protective immunity of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB, gC, gD and gE.

We have constructed recombinants of bovine herpesvirus-1 (BHV-1) which express pseudorabies virus (PRV) gB, gC, gD or gE either individually or in combination. To test the protective immunity, mice were inoculated with these BHV-1 recombinants and challenged three weeks later with virulent PRV. A BHV-1 recombinant, BHV-1/TF7-1, which express PRV gC, gD, gE and gI but not PRV gB, protected all 7 mice from the challenge with 20 LD50 virulent PRV and 6 out of 7 from the challenge with 100 LD50 PRV, while one dead mice survived for 5 days after the challenge. All the control mice died in 3 days. BHV-1 recombinants which express PRV gB, gC and gD individually also gave some protection but not so effective as BHV/TF7-1. Before challenging with virulent PRV, sera were collected from immunized mice and antibody against PRV was assayed. Western blot analyses indicated that all the recombinants induced antibody in mice against PRV gB, gC or gD individually or in combination. Virus neutralizing (VN) titer against PRV was highest in mice which were inoculated with BHV-1/TF6-1, which expresses PRV gB. BHV-1/TF7-1, which was more effective to protect mice from the challenge of virulent PRV, induced lower VN titer.

Animals↗

PubMed2005

[The expression of recombinant protein of human interleukin-24 and its anti-tumor mechanism].

The hIL24 cDNA sequence was cloned into prokaryotic high expressive vector pET-21a(+) and recombinant hIL24 was expressed in E. coli with IPTG induction. The purified recombinant hIL24 exhibits following functions in HeLa cell: inhibiting cell growth, inducing apoptosis, inducing PMBC to secrete IL-6, TNF-alpha, IFN-r and inhibiting blood vessel formation. Our preliminary results suggest that the apoptosis induced by rhIL24 is through down-regulating expression of anti-apoptosis factor Bcl-2 and activation of mitochondria apoptosis pathway.

Apoptosis↗

PubMed1991

Protection of mice with vaccinia virus recombinants that express the rabies nucleoprotein.

The role of rabies virus nucleoprotein (N) in protection against rabies was examined with recombinant vaccinia viruses expressing the N of the Challenge Virus Standard strain. Two chimeric plasmids were constructed with the open reading frame of the N gene placed downstream of the vaccinia P7.5 promoter (early/late class) or the vaccinia P11 promoter (late class), with each expression cassette flanked by vaccinia thymidine kinase (TK) sequences to enable marker rescue by TK insertional inactivation. Two recombinants were isolated that expressed the rabies N in infected cells as determined by radioimmunoprecipitation and immunofluoresence microscopy with an anti-N monoclonal antibody. Two groups of 25 ICR mice inoculated intradermally with the recombinants and challenged with 75 MFPLD50 of street rabies virus showed high survival ratios (22/25 and 21/25). Intramuscular inoculation, however, was not protective against 25 MFPLD50. The intradermally vaccinated mice developed non-neutralizing antibodies against rabies N.

Animals↗

PubMedDate not supplied

HEK293 cell line: a vehicle for the expression of recombinant proteins.

The HEK cell line has been extensively used as an expression tool for recombinant proteins since it was generated over 25 years ago. Although of epithelial origin, its biochemical machinery is capable of carrying out most of the post-translational folding and processing required to generate functional, mature protein from a wide spectrum of both mammalian and non-mammalian nucleic acids. Though popular as a transient expression system, this cell type has also seen wide use in stably transfected forms (i.e. transformed cells) to study a variety of cell-biological questions in neurobiology. The principal attributes which have made the HEK cell a popular choice among electrophysiologists to study isolated receptor channels include; its quick and easy reproduction and maintenance; amenability to transfection using a wide variety of methods; high efficiency of transfection and protein production; faithful translation and processing of proteins; and small cell size with minimal processes appropriate for voltage-clamp experimentation. These, and other attributes, also mean that complementary biochemical/cell biological evaluations of expressed proteins can be performed in concert with functional analyses to establish detailed pharmacological and biophysical profiles for the action of new drugs and their targets. The increased amount of sequence information available from the human genome has placed greater emphasis upon heterologous cell expression systems as targets for high throughput structure-function evaluation of novel drug targets and disease markers. Here we have highlighted some of the innate characteristics of the HEK cell in order that its suitability as a vehicle for the expression of a gene product can be assessed for particular needs. We have also detailed some of the standard methods used for transfection and obtaining functional data from electrophysiological recording techniques.

Cell Line↗

PubMed2003

[Feeding of mixed-carbon-resource during the expression phase in cultivation of recombinant Pichia pastoris expressing angiostatin].

A recombinant strain of Pichia pastoris with a phenotype of Muts was used to produce angiostatin in a 5-L fermentor. The methanol utilization ability of the present strain was weak, which resulted in extremely low growth rate and angiostatin productivity during the expression phase with methanol as the sole carbon source. To enhance the cell density and angiostatin expression level, mixed-carbon-source of glycerol-methanol was used in the expression phase. The methanol concentration was well controlled at 5 g/L by a methanol sensor and control system, and glycerol was continuously fed into the fermentor to achieve a higher cell density. 120 g/L of cells and 39 mg/L of angiostatin were reached at the end of fermentation which lasted 110 h. The mean specific cell growth rate in the expression phase was 0.01 h(-1), and the mean specific angiostatin productivity was 0.006 mg/(g x h). According to the data obtained in several runs of fermentation in which glycerol was fed at different rates, a higher mean specific angiostatin productivity was reached at the mean specific cell growth rate of 0.012 h(-1). To avoid the repression of angiostatin expression caused by residual glycerol and ethanol accumulation due to overfeeding of glycerol, glycerol addition was controlled to produce continuous oscillations in dissolved oxygen, because the change of dissolved oxygen concentration could deliver the information of available carbon source in the fermentation broth. Controlled glycerol feeding also avoided the problem of oxygen limitation brought by high cell density, and thus decreased the cooling requirement of the fermentor. Cell density reached 150 g/L at the end of fermentation, and angiostatin level reached 108 mg/L after an expression period of 96 h when the mean specific growth rate was maintained at 0.012 h(-1) by using the glycerol feeding strategy to result in the oscillations in dissolved oxygen. The mean specific angiostatin productivity was improved to 0.02 mg/(g x h). The apparent cell yield on glycerol and methanol were respectively 0.69 g/g and 0.93 g/g, higher than those in the fermentation without using the feeding strategy with dissolved oxygen as the indicator of metabolism.

Angiostatins↗

PubMed2001

Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus.

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap.

Adenoviridae↗

PubMed2000

Expression of recombinant human type I-III collagens in the yeast pichia pastoris.

An efficient expression system for recombinant human collagens will have numerous scientific and medical applications. However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity. We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methylotrophic yeast Pichia pastoris. These methods are based on co-expression of procollagen polypeptide chains with the alpha- and beta-subunits of prolyl 4-hydroxylase. The triple-helical type-I, -II and-III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins. All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils. The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g/l depending on the collagen type.

Amino Acid Sequence↗

PubMed2002

Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily.

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by recombinant yeast cells expressing either rat CYP1A1 or CYP1A2 was examined. When each of the dibenzo-p-dioxins (DDs), mono-, di-, and tri-chloroDDs, was added to the cell culture of the recombinant yeast, a remarkable metabolism was observed. The metabolism contained multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and opening of dioxin ring. The distinct difference was observed in substrate specificity and reaction specificity between CYP1A1 and CYP1A2. Kinetic analysis using microsomal fractions prepared from the recombinant yeast cells revealed that 2,7-dichloroDD and 2,3,7-trichloroDD were good substrates for both CYP1A1 and CYP1A2. When 2,3,7-trichloroDD was added to the yeast cells expressing each of rat CYP1A1 and CYP1A2, most of 2,3,7-trichloroDD was first converted to 8-hydroxy-2,3,7-trichloroDD, and further metabolized to more hydrophilic compounds whose ethereal bridges were cleaved. These findings give essential information on the metabolism of PCDDs in mammalian liver. In addition, this study indicates the possibility of application of microorganisms expressing mammalian cytochrome P450 to bioremediation of contaminated soils with dioxins.

Animals↗

PubMed2003

Expression of recombinant proteins in a lipid A mutant of Escherichia coli BL21 with a strongly reduced capacity to induce dendritic cell activation and maturation.

Mutations in the Escherichia coli (E. coli) and Salmonella lpxM gene have been shown to result in strains which grow normally and which produce a non-myristoylated lipopolysaccharide (nmLPS) with strongly reduced endotoxicity. Using homologous recombination, we inactivated the lpxM gene in BL21 (DE3), a strain widely used for the production of recombinant proteins. This led to a derivative unaffected in its capacity to support the production of recombinant proteins. This new strain expresses non-myristoylated LPS that induces markedly less activation and maturation of monocyte-derived dendritic cells (DC), as assessed by nuclear translocation of nuclear factor kappa B (NF-kappaB), production of TNF-alpha and IL-8 or expression of CD86. Activation of the main signal transducing receptor for extracellular LPS, Toll like receptor (TLR) 4 in conjunction with the soluble accessory protein MD-2 was also markedly decreased. The modified BL21 strain represents a new application of lpxM inactivation for the expression of proteins to be tested on dendritic cells or other LPS sensitive cells/receptor complexes. It is likely to be useful for the identification of new proteins activating the innate immune response and to reducing the risk linked with low level of endotoxin contamination in therapeutic recombinant proteins.

Antigens, CD↗

PubMed1986

Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli.

The high-level expression of human interleukin-1 beta in Escherichia coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion- and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N- and C-terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vitro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1/LAF) assay. The specific activities determined with the IL-1/MCF and IL-1/LAF assays, are 2 X 10(7) and 4 X 10(7) units mg-1, respectively.

Amino Acid Sequence↗

PubMedDate not supplied

High concentrations of recombinant adenovirus expressing p16 gene induces apoptosis in lung cancer cell lines.

In this study, we discussed the effects of treatment with recombinant adenovirus expressing p16 (AX-p16) on cell growth and cell death. Ax-p16 at 10 m.o.i. groups showed growth inhibition 3 days after gene transfection, but the cells regrew and did not undergo cell death. On the other hand, Ax-p16 at 300 m.o.i. groups showed complete cell growth inhibition leading to cell death which was apparent 7 days after p16 gene transfection. In the high m.o.i. Ax-mock groups, cell death was marked just after infection, but had diminished by 7 days after infection. Downregulation of pRB was detected only in Ax-p16 at 300 m.o.i. groups. These data suggest that a) high m.o.i. condition of Ax-p16 gives therapeutic benefits due to the combined effects of adenovirus and high expression of p16; and b) the cell killing mechanism of the p16 transgene is different from that of high m.o.i. adenoviral infection.

Adenoviruses, Human↗

PubMed1999

A rapid and safe plasmid isolation method for efficient engineering of recombinant lactobacilli expressing immunogenic or tolerogenic epitopes for oral administration.

Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications. Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated from Lactobacillus casei to transform other Lactobacillus strains. The isolation of plasmid DNA from Gram-positive lactobacilli is complicated by the resilience of the peptidoglycan layer. Here a rapid, safe and efficient method is described that combines enzymatic breakdown of the cell wall and purification of the plasmid by commercially available DNA-binding columns. For the lysis-resistant L. casei strain, this method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency.

Administration, Oral↗

PubMedDate not supplied

[A recombinant adenovirus expressing the NS1 nonstructural protein of tick-borne encephalitis virus: some characteristics of the immunologic basis of antiviral action].

Recombinant adenovirus expressing NS1 nonstructural protein of trick-borne encephalitis (TBE) virus (Rad 51) protected mice from many strains of TBE and Omsk hemorhagic fever (OHF) viruses, but virtually did not protect them from Negishi virus. During combined use of whole-virion inactivated TBE vaccine and Rad 51 the recombinant adenovirus notably potentiated the protective effect of the traditional vaccine. The results of adaptive transfer of immunological material from mice infected with Rad 51 showed that both the vaccinated animals' sera and the pool of T and B cells partially protected the recipient mice from lethal TBE infection. NS1 protein expressed by adenovirus increased the level of the key interleukins (IL) interferon, tumor necrosis factor, IL-1 beta, IL-2, and, probably, IL-4. Vaccination of mice with Rad 51 resulted in the appearance of antibodies to NS1 protein in rather high titers. The prospects of using Rad 51 as a vaccine against TBE are discussed.

Adenoviridae↗