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PubMed2004

Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins.

For structural and functional genomics programs, new high-throughput methods to characterize well-expressing and highly soluble proteins are essential. A faster and more convenient approach to screen expression conditions of recombinant proteins compared to classical in vivo systems is the Escherichia coli cell-free expression system. Here, we describe a rapid procedure to screen for expression and solubility of recombinant proteins using an E. coli cell-free extract. The results presented cover 24 open reading frames of unknown function from different micro-organisms. In order to screen different variables that may interfere with solubility, we expressed the recombinant proteins with a histidine6 tag, either N-terminal or C-terminal at two temperatures (25 degrees C and 30 degrees C). The identification of recombinant proteins is performed by the dot blot procedure using an anti-histidine tag antibody. We designed a rapid method that allows the characterization of soluble candidates from a large number of genes or from a large number of variants that is highly compatible with structural genomics expectations.

DNA, Recombinant↗

PubMed1994

Formation of sulphmyoglobin during expression of horse heart myoglobin in Escherichia coli.

Expression of recombinant horse heart myoglobin in Escherichia coli has been found to result in the production of both native and variable amounts (approximately 16-17% total) of two sulphmyoglobin isomers. The recombinant sulphmyoglobin produced consists primarily of the A and B isomers as identified by 1H NMR spectroscopy with no evidence for production of the C isomer. Conversion of recombinant sulphmyoglobin to the native protein can be achieved by reconstitution with protohaem IX. The possible relationship of this observation to recombinant expression of other heme proteins is discussed.

Animals↗

PubMed1992

Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line.

Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.

Amino Acid Sequence↗

PubMed2004

Expression of recombinant endostatin in Agrobacterium-inoculated leaf disks of Nicotiana tabacum var. Xanthi.

Recombinant endostatin was transiently expressed in Agrobacterium-inoculated leaf disks of Nicotiana tabacum var. Xanthi with a molecular size of 23 kDa. Expression of endostatin from a replicating vector based on tomato golden mosaic virus (TGMV) was 170% higher at the transcript level and double higher at the protein level than from a control vector of a non-replicating construct. Purified recombinant endostatin from tobacco leaf-disks has an anti-proliferative effect on bovine endothelial cells.

Animals↗

PubMed1987

Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses.

A complete set of recombinant vaccinia viruses that express each of the influenza virus polypeptides has been constructed. PB1, PB2, PA, HA, NP, M1, and NS1 genes were derived from influenza virus A/PR/8/34, NA from influenza virus A/Cam/46, and M2 and NS2 genes from influenza virus A/Udorn/72. Cells infected with these recombinant viruses synthesize influenza polypeptides that are precipitable with specific antisera and that have electrophoretic mobilities similar to the corresponding influenza virus polypeptides. Indirect immunofluorescence studies have shown that HA, NA, and MS2 proteins migrate to the cell surface; PB2, PB1, PA, NP, and NS1 proteins migrate to the cell nucleus; and M1 and NS2 are distributed throughout the cell, although NS2 accumulates preferentially in nuclei. These transport processes occurred independently of other influenza polypeptides and are therefore attributable to the intrinsic properties of the influenza polypeptides themselves.

Antigens, Viral↗

PubMed2001

Silent mutations in secondary Shine-Dalgarno sequences in the cDNA of human serum amyloid A4 promotes expression of recombinant protein in Escherichia coli.

The serum amyloid A (SAA) superfamily comprises a number of differentially expressed genes with a high degree of homology in mammalian species. SAA4, an apolipoprotein constitutively expressed only in humans and mice, is associated almost entirely with lipoproteins of the high-density range. The presence of SAA4 mRNA and protein in macrophage-derived foam cells of coronary and carotid arteries suggested a specific role of human SAA4 during inflammation including atherosclerosis. Here we underline the importance of ribosome binding site (rbs)-like sequences (also known as Shine-Dalgarno sequences) in the SAA4 cDNA for expression of recombinant SAA4 protein in Escherichia coli. In contrast to rbs sequences coded by the expression vectors, rbs-like sequences in the cDNA of target protein(s) are known to interfere with protein translation via binding to the small 16S ribosome subunit, yielding low or even no expression. Here we show that PCR mutations of two rbs-like sequences in the human SAA4 cDNA promote expression of considerable amounts of recombinant SAA4 in E.coli.

Binding Sites↗

PubMed1992

Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus.

An expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram-positive bacterium, is described. The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. The expression system was used to secrete fusion proteins containing sequences from a malaria blood-stage antigen and a streptococcal albumin-binding receptor to the cell surface of S. xylosus. Analysis of the recombinant cells by immunogold staining and immunofluorescence revealed that both the receptor and the malaria peptide were properly processed and exposed on the surface of the host cells. However, only approximately 40 to 50% of the recombinant cells were strongly stained with antiserum reactive with the albumin-binding receptor, while approximately 10 to 15% of the cells were stained with antiserum reactive with the malaria peptide. The incomplete staining of some of the cells suggests steric effects that make the recombinant fusion proteins inaccessible to the reactive antibodies because of variable cell wall structures. However, the results demonstrate for the first time that recombinant techniques can be used to express heterologous receptors and immunogens on the surface of gram-positive cells.

Amino Acid Sequence↗

PubMed2002

Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27(Kip1).

BACKGROUND: Adp27(Kip1), a recombinant adenovirus, was evaluated for expression of p27, a cyclin-dependent kinase inhibitor (CDKI) and tumor suppressor protein, in human prostate carcinoma cells. Effects of p27(Kip1) on cell cycle and apoptosis were analyzed. METHODS: We evaluated the effects of overexpression of p27(Kip1) in the human prostate carcinoma cell lines LNCaP, DU-145, and PC-3 in vitro and in vivo. Growth curve studies, cell cycle analysis, terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL), and annexin V-fluorescein isothiocyanate apoptosis analyses were conducted to determine effects of p27(Kip1) on cell cycle. CDKI activity assays and Western blots were conducted to determine presence/activities of CDKIs. RESULTS: Adp27(Kip1)-induced protein levels increased in a dose-dependent manner; p27(Kip1) protein was detected within 6 hr of infection with Adp27(Kip1) and remained stable for at least 48 hr. The activities of Cdk2, Cdk4, and Cdc2 kinases were inhibited 24 hr after infection with Adp27(Kip1). Bromodeoxyuridine incorporation demonstrated a dose-dependent decrease in S-phase cells 24 hr postinfection. TUNEL analysis revealed an induction of apoptosis (10 pfu/cell) within 48 hr of infection in all cell lines. Growth curve analyses demonstrated that Adp27(Kip1) infection inhibited proliferation of all cell lines tested and decreased cell numbers for Adp27(Kip1)-infected LNCaP and PC-3 cells by 96 hr. Cell cycle analysis of DNA content demonstrated an accumulation of cells in G0/G1-phase 24-120 hr after Adp27(Kip1)-infection. In vivo studies demonstrated a reduction in LNCaP xenograft tumor growth rates in mice injected with Adp27(Kip1). CONCLUSION: Exogenous p27(Kip1) overexpression results in cell cycle regulation in the human prostate carcinoma cell lines tested, representing the first use of this vector on prostate cancer cell lines in vitro and in vivo. Moreover, p27(Kip1) expression is associated with an increase in early apoptosis, which represents a recently discovered function for this protein. It also represents the first time this association has been observed in prostate carcinoma cell lines. This study provides support for the further development of Adp27(Kip1) as a potential therapeutic vector in the treatment of adenocarcinoma of the prostate.

Adenoviruses, Human↗

PubMed1999

The refolding, purification, and activity analysis of a rice Bowman-Birk inhibitor expressed in Escherichia coli.

A putative rice trypsin/chymotrypsin inhibitor of the Bowman-Birk family, RBBI-8 of about 20 kDa, was expressed in Escherichia coli as a fusion protein bearing an N-terminal (His)6 purification tag. The expressed recombinant protein, rRBBI-8, is insoluble and accumulates as inclusion bodies. The insoluble protein was solubilized in 8 M urea under reducing environment and then refolded into its active conformation under optimized redox conditions. Strategies used to optimize yield and efficiency include selecting the redox system, increasing protein concentration during refolding by adding the denatured protein in a stepwise way, utilizing additives to prevent aggregation, and selecting buffer-exchanging conditions. A Ni-chelate affinity column was then employed to purify the renatured protein. rRBBI-8 shows strong inhibitory activity against trypsin and it can slightly inhibit chymotrypsin. In this study, a refolding and purification system was set up for this cysteine-rich recombinant protein expressed in a prokaryotic system.

Amino Acid Sequence↗

PubMed2005

Construction of recombinant eukaryotic expression plasmid containing murine CD40 ligand gene and its expression in H22 cells.

AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC). METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing, and the product expressed was detected by RT-PCR and immunofluorescence methods. RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis, and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immunofluorescence staining. CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

Animals↗

PubMed1991

Alzheimer's amyloid precursor protein produced by recombinant baculovirus expression. Proteolytic processing and protease inhibitory properties.

The baculovirus expression system was used to generate recombinant Alzheimer's amyloid precursor (AAP) proteins. Recombinant baculoviruses were constructed, designed to express full-length 695-, 751-, and 770-amino acid forms. Recombinant baculoviruses designed for constitutive secretion were engineered by placing a termination codon between the beta-protein domain and cytoplasmic anchor of the full-length forms. Insect cells infected with each of these baculoviruses produced both secreted and cell-associated AAPs. Full-length constructs produced secreted derivatives which were COOH-terminally cleaved within the beta-protein domain at Gln15 or Lys16, essentially identical to previous reports utilizing mammalian cell systems. Rare secreted forms (less than 5%) appeared to extend to Lys28. Secretion constructs produced these same forms, but in different ratios. Most (approximately 60%) terminated at Gln15 or Lys16, while the remainder apparently extended to Lys28. AAPs containing the Kunitz-type serine protease inhibitory domain (AAP-751 and -770) were shown to be active inhibitors. No differences were observed in the inhibitors activities of these two forms. The similarities in AAP processing by insect and mammalian systems, together with the large amounts of recombinant protein produced by baculovirus expression, make this an attractive system for studies of AAP processing and biochemical properties.

Alzheimer Disease↗

PubMed2000

Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escherichia coli.

The affinity of natural antibody (Ka = 8 x 10(6) M(-1)) recognizing preS1 of hepatitis B virus (HBV) was improved by replacing the heavy (H) chain gene with repertoires of VH genes, obtained from two nonimmunized donors. Two separate clones, 1C2 and 1E4, showed affinities of 2.3 x 10(7) and 5.2 x 10(7) M(-1), which were increased by factors of 2.8 and 6.5, respectively, compared to the parental clone. Recombinant scFvs (rscFvs) were expressed as fusion protein with minor coat protein, pIII, and secreted into medium after 3 h of induction with 1 mM IPTG. The expression level of functional rscFv capable of binding to preS1 reached a peak after 6-10 h (1C2) and 8-10 h (1E4) of IPTG induction, and afterwards decreased gradually. In order to achieve the overexpression of rscFv in E. coli, gene encoding scFv of 1C2 or 1E4 was inserted into pRSET vector. RscFvs were overexpressed as cytoplasmic inclusion bodies in E. coli BL 21 strain, which were denatured and carefully refolded using a continuous dialysis system. The purified recombinant fragments were pure when analyzed by SDS-PAGE and had the predicted size of 34 kDa. Clone 1E4 used the heavy chain gene belonging to family VII and subgroup III. Chain shuffling offers an alternative to random point mutation for affinity maturation of human antibody in vitro.

Amino Acid Sequence↗

PubMed2003

FT-IR study of heterologous protein expression in recombinant Escherichia coli strains.

Two recombinant Escherichia coli strains expressing different levels of an interferon fusion protein as inclusion bodies have been studied by Fourier transform infrared (FT-IR) microspectroscopy. A marker band at 1628 cm(-1) allowed monitoring of the protein expression by direct analysis of cell pellets in a rapid, non-invasive and quantitative way. The results demonstrate that FT-IR microspectroscopy is a technique of potential biotechnological interest for studying inclusion body formation.

Escherichia coli↗

PubMed1996

Heterogeneous expression of recombination activating genes and surface CD5 in CD3low CD4+ CD8+ thymocytes.

Clonal selection in the thymus occurs mostly in the CD4+ CD8+ (double positive; DP) stage. Within DP thymocytes, cells in the CD3low subset are believed to be involved in clonal selection, and this subset is generally considered as a homogeneous population. T-cell antigen-receptor (TCR) signals on DP thymocytes are known to (i) down-regulate recombination activating gene (RAG) expression; and (ii) up-regulate the expression of T-cell function-associated molecules, including the cell surface glycoprotein CD5. The present study examined the expression of RAG and CD5 molecules among the subpopulations of normal adult DP thymocytes. DP thymocytes were fractionated according to their cell surface expression levels of TCR-CD3 complex into CD3dim, CD3lo, CD3med, and CD3hi cells, since TCR expression is known to increase during thymocyte maturation. Down-regulation of RAG mRNA was located between the CD3low and CD3med DP populations. However, within the DP CD3low subpopulation, we found that CD5 varies from low to high expression levels. Upon fractionation of DP CD3low thymocytes into CD5low and CD5high subsets, we were able to detect down-regulation of RAG transcripts within the CD3low subpopulation. Thus, by the criteria of CD5 surface expression and RAG mRNA expression levels, DP CD3low thymocytes can be considered a heterogeneous thymocyte subpopulation.

Animals↗

PubMed1992

The basal promoter elements of murine cytochrome c oxidase subunit IV gene consist of tandemly duplicated ets motifs that bind to GABP-related transcription factors.

DNA sequences required for expression of the mouse cytochrome c oxidase subunit IV (COXIV) promoter were identified by transient expression of recombinant COXIV-chloramphenicol acetyltransferase constructs in COS and NIH-3T3 cells. Activity of the COXIV promoter is shown to depend upon upstream Sp1 binding sequences and two tandemly repeated 21-base pair sequence elements each mapping to sites of mRNA initiation. Each initiation region repeat contains a binding site for an ets-related transcription factor which demonstrates specificity for the characteristic GGAA ets sequence motif and reactivity with an ets domain-directed monoclonal pan ets antibody. The two 21-base pair repeats are sufficient for transcriptional activity suggesting that the ets-related factor may be involved in both transcriptional activation and start site positioning. The ets-related protein found in COS nuclear extracts is shown to be identical or closely related to the GA-binding protein (GABP) by comparison of electrophoretic mobilities and immunological reactivities of DNA-protein complexes formed with purified recombinant expressed GABP alpha and beta subunits. Sp1 and the GABP-related factors also bind to another mouse cytochrome oxidase subunit gene COXVb. The similar promoter features of these two genes suggests a possible means of coordinate transcriptional regulation among such respiratory proteins.

3T3 Cells↗

PubMedDate not supplied

Efficient selection of stable chinese hamster ovary (CHO) cell lines for expression of recombinant proteins by using human interferon beta SAR element.

We describe the development of an efficient expression system suitable for the stable expression of recombinant genes in Chinese hamster ovary (CHO) cells using the human interferon beta SAR element. The insertion of two copies of the human interferon beta SAR element at the 5' and 3' flanking regions of the beta-galactosidase reporter gene increased the frequency of beta-galactosidase positive colonies by up to 75% and enhanced beta-galactosidase expression by 15- to 20-fold after G418 selection or 30- to 40-fold at the initial stage of the MTX selection procedure. Deletion analysis showed that the whole DNA regions of the human interferon beta SAR element are required for beta-galactosidase expression enhancement. The developed expression system was also highly effective at enhancing the stable expression of two therapeutically important proteins, namely, erythropoietin (EPO) and hepatocyte growth factor (HGF). We isolated stable colonies with expression levels of 47 microg/10(6) cells/day for EPO and 13 microg/10(6) cells/day for HGF, suggesting that the developed expression system based on the human beta SAR element is suitable for expressing high levels of recombinant proteins in CHO cells.

Animals↗

PubMed1987

Efficient and stable expression of recombinant fibronectin polypeptides.

We describe retroviral expression vectors containing cDNAs encoding part of fibronectin preceded by the signal and "pro" sequences of parathyroid hormone. The recombinant retroviruses were used to generate NIH 3T3 cell lines stably producing functionally active fragments of fibronectin. The recombinant fibronectins (deminectins) are processed and secreted by the cells and form disulfide-bonded dimers with themselves and with endogenous fibronectin subunits. The fibronectin-deminectin heterodimers are incorporated into the extracellular matrix. We describe cell lines producing six variant forms of deminectin corresponding to variant forms of fibronectin produced by alternative splicing. In constructing fibronectin cDNAs encoding the six variant forms, we also made use of the ability of retroviral vectors to generate cDNAs by accurate splicing of cloned genomic segments. These constructs should be valuable in analyses of the structure-function relationships of fibronectins.

Animals↗

PubMed2005

Expression, refolding, and purification of recombinant human granzyme B.

Granzyme B (GrB) is a member of a family of serine proteases involved in cytotoxic T-lymphocyte-mediated killing of potentially harmful cells, where GrB induces apoptosis by cleavage of a limited number of substrates. To investigate the suitability of GrB as an enzyme for specific fusion protein cleavage, two derivatives of human GrB, one dependent on blood coagulation factor Xa (FXa) cleavage for activation and one engineered to be self-activating, were recombinantly expressed in Escherichia coli. Both derivatives contain a hexa-histidine affinity tag fused to the C-terminus and expressed as inclusion bodies. These were isolated and solubilized in guanidiniumHCl, immobilized on a Ni2+-NTA agarose column, and refolded by application of a cyclic refolding protocol. The refolded pro-rGrB-H6 could be converted to a fully active form by cleavage with FXa or, for pro(IEPD)-rGrB-H6, by autocatalytic processing during the final purification step. A self-activating derivative in which the unpaired cysteine of human GrB was substituted with phenylalanine was also prepared. Both rGrB-H6 and the C228F mutant were found to be highly specific and efficient processing enzymes for the cleavage of fusion proteins, as demonstrated by cleavage of fusion proteins containing the IEPD recognition sequence of GrB.

Cloning, Molecular↗