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PubMed2005

A quantitative slot blot assay for host cell protein impurities in recombinant proteins expressed in E. coli.

Residual host cell protein impurities in recombinant proteins intended for human use must be accurately quantified to help establish their safety. We describe a novel means of host cell protein quantitation, in which a slot blot system was employed together with scanning laser densitometry to allow picogram level sensitivity in detection of residual host cell proteins in unpurified fermentation products and final purified bulk samples. Two allelic forms of merozoite surface protein 1, a promising malaria vaccine candidate antigen currently undergoing evaluation in clinical trials, were expressed in E. coli as clinical grade proteins, refolded, and carried through several chromatographic purification steps. Several lots of these proteins were analyzed with this generic quantitative assay that uses rat polyclonal antibodies generated against soluble and insoluble E. coli proteins. The assay had a detection range of 6.1-1562 ng/mL, with a detection limit of 6.1 ng/mL, comparable to reported ELISA-based methods. This assay proved simple yet very sensitive and accurate, giving highly reproducible results. Thus it is suitable for evaluating host cell protein levels in clinical grade recombinant proteins expressed in E. coli.

Animals↗

PubMed1996

The expression of recombinant proteins in yeasts.

The methylotrophic yeasts Hansenula polymorpha and Pichia pastoris are rapidly becoming the systems of choice for the expression of recombinant proteins in yeast. However, the powerful genetic techniques available in Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are still exploited to establish models to study medically important cell processes and screen for pharmacologically active compounds.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Expression of recombinant small hydrophobic protein for serospecific detection of avian pneumovirus subgroup C.

The small hydrophobic (SH) gene of the avian pneumovirus (APV) Colorado isolate (CO), which belongs to subgroup C (APV/C), was expressed with a baculovirus vector. The recombinant SH protein was evaluated as a potential subgroup-specific diagnostic reagent in order to differentiate infections resulting from APV/C from those induced by APV/A, APV/B, and human metapneumovirus (hMPV). When the recombinant baculovirus was used to infect insect cells, a 31- to 38-kDa glycosylated form of the SH protein was produced and subsequently tested for reactivity with antibodies specific for APV/A, APV/B, APV/C, and hMPV. Western blot analysis showed that the expressed recombinant SH protein could only be recognized by APV/C-specific antibodies. This result was consistent with sequence analysis of the APV/C SH protein, which had very low (24%) amino acid identity with the corresponding protein of hMPV and no discernible identity with the SH protein of APV/A or APV/B. A recombinant SH protein-based enzyme-linked immunosorbent assay (ELISA) was developed, and it further confirmed the lack of reactivity of this protein with antisera raised to APV/A, APV/B, and hMPV and supported its designation as a subgroup-specific antigen. This finding indicated that the recombinant SH protein was a suitable antigen for ELISA-based detection of subgroup-specific antibodies in turkeys and could be used for serologically based differential diagnosis of APV and hMPV infections.

Amino Acid Sequence↗

PubMed1986

Expression of recombinant DNA functional products in Escherichia coli anucleate minicells.

This review covers the use of anucleate minicells of Escherichia coli for expressing the recombinant DNA encoded proteins. We briefly discuss the methods being used for preparation of anucleate minicells, incorporation of cloned DNA and assessment of gene expression. While the largest use has been that of microbially derived cloned functional DNA, examples of eukaryotic gene product synthesis have also been reviewed. This technology may represent some interesting commercial opportunities.

Journal Article↗

PubMed2000

[Expression of recombinant human stem cell factor in insect cells].

The cDNA of human stem cell factor(hSCF) containing signal sequence was cloned into the transfer vector pVL941 of AcNPV to construct a recombinant transfer vector pVL941-SCF. Sf9 cells were cotransfected with wild type viral DNA and pVL914-SCF to produce the recombinant virus AcNPV-SCF by homologous recombination in cell. Southern-hybridization analysis suggested that the recombinant viral DNA contained hSCF cDNA fragment. The Sf9 cells infected with the recombinant baculovirus AcNPV-SCF expressed biologically active rhSCF which was secreted into the cell culture. The synergistic activities of SCF in conjunction with human interleukin-3(hIL-3) was measured by MTT colorimetric method and TF-1 cell line. The expression level of Sf9 cells reached its highest at about 1970 units/ml in the 3rd day after the infection with AcNPV-SCF. Three SCF bands with molecular masses of 18 x 10(3), 20 x 10(3) and 22 x 10(3) were detected by immunoblotting.

Animals↗

PubMed1994

Double immunogold labelling demonstrating expression of recombinant genes for production of an anti-fertility vaccine.

The multiple antibody technique for double immunogold labelling for the simultaneous localization of two antigens with negative staining was utilized to demonstrate the expression of recombinant genes in bacteria, with the primary antibodies being raised in different host species. For the production of a vaccine for immunological control of fertility, a multi-functional plasmid vector was introduced into the bacterium Pseudomonas aeruginosa containing the Dichelobacter nodosus fimbrial subunit gene with a grafted amino acid sequence of luteinizing hormone releasing hormone (LHRH) peptide. Fimbriae of this recombinant, when run on a SDS-polyacrylamide electrophoresis gel, gave a single broad band for LHRH peptide/D. nodosus subunit and were harvested to produce the anti-fertility vaccine which, when injected into mice, produced atrophy of the testes with absence of sperm, resulting in reversible castration. Double immunogold labelling of the recombinant P. aeruginosa bacteria demonstrated fimbriae with strong expression of the LHRH-peptide, expression of the D. nodosus subunit and absence of host fimbriae.

Animals↗

PubMed1992

Function and immunogenicity of human parainfluenza virus 3 glycoproteins expressed by recombinant adenoviruses.

Human parainfluenza virus type 3 fusion (F) and hemagglutinin-neuraminidase (HN) cDNA sequences were inserted into the E3 region of the adenovirus type 5 genome. Cells infected with recombinant adenoviruses containing HPIV3 F (AdF) and HN (AdHN) sequences were shown to express HPIV3 F and HN proteins that were functional and immunogenic. The HN protein produced following AdHN infection was glycosylated, expressed on the surface of infected cells and exhibited both hemagglutinin and neuraminidase activities. AdF infection led to the synthesis of both the HPIV3 F0 precursor and its proteolytic cleavage product, F1. F proteins produced by AdF were glycosylated and expressed on the infected cell surface. Syncytium formation was observed in HeLa T4 cell monolayers upon coinfection with AdF and AdHN. The F and HN proteins expressed by recombinant adenoviruses were recognized by HPIV3 F- and HN-specific monoclonal antibodies. Mice injected intraperitoneally with AdF or AdHN produced antibodies that immunoprecipitated the appropriate HPIV3 glycoproteins and sera from immunized mice effectively neutralized HPIV3 virions. These results support future work using recombinant adenoviruses to study the immune response to individual HPIV3 glycoproteins as well as in protection studies using animal models.

Adenoviridae↗

PubMed1999

Expression of recombinant feline serum amyloid A (SAA) protein.

Feline serum amyloid A (SAA) cDNA clone was isolated from a hepatic mRNA of a mixed-breed cat. The feline SAA cDNA clone contains 333 nucleotides and deduced amino acid sequence shows 87.4%, 73.9%, and 65.8% homology with dog, human and mouse SAA respectively. Relative to the human and mouse SAA proteins, an additional peptide of eight amino acids is specified in the feline cDNA clone. Recombinant feline SAA (rfSAA) was expressed at high levels using pGEX bacterial expression system. Cleavage from the fusion moiety, and purification using glutathione-sepharose yielded pure soluble form of rfSAA. Antibodies generated against rfSAA were specific for feline SAA and showed no cross-reactivity with human SAA. Furthermore, antibodies against human SAA did not react with feline SAA. These results indicate that antigenicity of feline SAA is totally different from human SAA.

Amino Acid Sequence↗

PubMedDate not supplied

[Differential expression of recombinant hybrid proteins containing amino- and carboxy-terminal regions of the exterior large glycoprotein (gp46) of the human T-cell leukemia virus (HTLV-I) in Escherichia coli cells].

Plasmid clones capable of expressing a recombinant fusion proteins containing the anthranilate synthase of E. coli (TrpE) and different regions of gp46 HTLV-I were constructed on the basis of pATH-vectors. A high extent of TrpE-gp46 proteolytic degradation took place independently of the bacterial La-protease. Fusion proteins containing an N-terminal part of gp46 were more stable and could be purified in preparative quantities but were less antigenic. On the contrary, a TrpE-gp46 protein encoded by the TaqI-TaqI DNA fragment and containing only 35 C-terminal amino acids was still susceptible to degradation but possessed good serologic reactivity. Some of the recombinant proteins obtained can be useful for diagnostics and for preparing monoclonal or polyclonal antibodies.

Anthranilate Synthase↗

PubMed1993

Expression of recombinant human thyroid peroxidase by the baculovirus system and its use in ELISA screening for diagnosis of autoimmune thyroid disease.

The cDNAs coding for human full-length and soluble thyroid peroxidase (TPO) were constructed, cloned into a baculovirus transfer vector and used for infection of Spodoptera frugiperda (Sf9) cells. The soluble TPO lacking 87 amino acids of the C-terminal transmembrane and intracisternal domains was designed as a fusion protein with a histidine-hexapeptide as an affinity ligand at its C-terminus. Whereas the recombinant full-length TPO was expressed mainly in an insoluble form in Sf9 cells, the recombinant soluble TPO was almost completely secreted into the culture medium. Both the full-length and the soluble TPO were purified by conventional methods and by a specific affinity chromatography using metal chelating matrix respectively, and tested for their autoantigenicity towards anti-TPO autoantibodies. The ELISA established with the purified recombinant soluble TPO as antigen demonstrated its specificity, practicability and reproducibility in screening of anti-TPO autoantibodies in sera of autoimmune thyroid patients. High correlation (r = 0.89, n = 175) was obtained between the soluble TPO and natural TPO prepared from human thyroid glands. Pathological sera (n = 200) were positively assayed with a significance of 91%.

Animals↗

PubMed2002

Immunotherapy for murine K1735 melanoma: combinatorial use of recombinant adenovirus expressing CD40L and other immunomodulators.

We have constructed and tested five recombinant adenoviruses (Ads) that express a variety of immunomodulators, including CD40 ligand (CD40L), a potent costimulator of several components of the immune system. We demonstrate that CD40L expressed from Ad in K1735 mouse melanoma cells leads to a strong reduction in tumorigenicity and to efficient protective immunity in a vaccination setting. Subsequently, using a therapeutic approach, we found that local, intratumoral coinjection of CD40L- and IL-2-expressing Ads was superior to any other agents tested and resulted in an at least 1.9-fold increase in mean survival time, in contrast to systemic application of recombinant CD40L or GM-CSF proteins, which had no significant effects. When using vaccination as a therapeutic approach, the combinations of CD40L plus IL-2 or GM-CSF plus IL-2 from Ad gave rise to an extended (2.8-fold) increase in mean survival time. A detailed analysis of immune cells present within regressing tumors indicated that mainly CD4(+) and CD8(+) T cells, and to a lesser extent dendritic cells, infiltrated the tumor mass, but not NK cells, macrophages, or granulocytes. These results propose that a combination of CD40L plus IL-2 has an improved efficacy over the use of single agents when applied for direct in situ therapy or vaccination therapy.

Adenoviridae↗

PubMed1988

Role of GM-CSF in phagocyte recruitment and regulation of c-fos protooncogene expression.

Recombinant GM-CSF induced migration across polycarbonate filters of monocytes and polymorphonuclear leukocytes. Checkerboard analysis revealed that induction of phagocyte migration involved actual chemotaxis. Hence GM-CSF, released by activated endothelium and leukocytes, may play a role in phagocyte recruitment to amplify resistance against noxious agents. Activation of leukocytes by GM-CSF was associated with early, transient augmentation of the expression of the c-fos protooncogene. c-fos expression may be associated with functional reprogramming of phagocytes by this cytokine.

Chemotaxis, Leukocyte↗

PubMed1997

Effect of recombinant human IL-4 on tryptase, chymase, and Fc epsilon receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells.

The effect of recombinant human IL-4 (rhIL-4) on the development of recombinant human stem cell factor-dependent fetal liver-derived mast cells was examined. RhIL-4 attenuates the number of mast cells that develop, preferentially affecting the MC(T) type of mast cell. Cellular levels of tryptase and chymase mRNA normalized to that of glyceraldehyde-3-phosphate dehydrogenase were not appreciably affected. Tryptase mRNA levels peaked at least 2 wk before tryptase protein and before chymase mRNA and protein, indicating that tryptase mRNA expression is an early marker of commitment to a mast cell lineage. In contrast, alpha-tryptase and beta-tryptase mRNA levels increased and decreased in parallel. The most dramatic effect of rhIL-4 was to induce expression of functional surface Fc epsilonRI. Expression was maximal by 21 days with 20 ng/ml of rhIL-4 and reached a plateau by 2 ng/ml of rhIL-4 at 4 wk. Fc epsilonRI+ cells increased modestly when myeloma IgE was added to the developing mast cells, but increased synergistically when both myeloma IgE and rhIL-4 were present together. Delayed addition of rhIL-4 progressively diminished Fc epsilonRI expression, as did withdrawal of rhIL-4 during the first 2 wk of culture. RhIL-4 selectively increased Fc epsilonRI alpha mRNA levels at least 10-fold. Mast cells developed in the presence of rhIL-4 released tryptase when exposed to anti-Fc epsilonRI alpha. In conclusion, induction of functional Fc epislonRI on recombinant human stem cell factor-dependent human fetal liver-derived mast cells by rhIL-4 harmonizes with the well-accepted ability of this cytokine to enhance IgE production by B cells.

Cells, Cultured↗

PubMed2005

Characterization of different cell culture media for expression of recombinant antibodies in mammalian cells: Presence of contaminating bovine antibodies.

Several new cell culture media designed specifically for the expression of recombinant antibodies in Chinese hamster ovary (CHO) cells were investigated for the presence of bovine IgG. Three serum-free media, three protein-free (animal component free) media, as well as one chemically defined medium were included in the study. Employing a combination of affinity chromatography (Protein G or A columns), SDS-PAGE analysis, and peptide mass fingerprinting, two of the serum-free media were found to contain bovine IgG in the range of approximately 0.5 mg/L. The other five media did not contain detectable levels of contaminating Protein A or G-binding proteins such as bovine IgG.

Animals↗

PubMed1992

Processing of hepatitis B virus surface antigen expressed by recombinant Oka varicella vaccine virus.

We have constructed a recombinant Oka varicella vaccine virus expressing hepatitis B virus (HBV) surface antigen (HBsAg). HBsAg was synthesized as 26K and 30K proteins in infected cells and secreted into the culture supernatant as 30K and 35K proteins. Inhibitors and glycosidase treatments, and pulse-chase labelling experiments, revealed the glycosylation process of HBsAg. The latter was synthesized as a non-glycosylated 26K protein and subjected to N-linked glycosylation to form a 30K protein with high mannose glycans. Three species of dimers composed of 26K and 30K subunits were then formed with disulphide bonds. Both subunits of the dimers were further subjected to O-linked glycosylation and conversion from high mannose glycans to complex glycans followed by sialylation. Three species of dimers composed of 30K and 35K subunits were secreted into the culture supernatant as HBsAg particles. HBsAg was synthesized, glycosylated with both N- and O-linked glycans, sialylated, and then secreted into the culture supernatant within 1 h. These modifications of HBsAg by glycans might stabilize its structure and enhance its immunogenicity as a live HBV vaccine.

Electrophoresis, Polyacrylamide Gel↗

PubMed1992

S100P, a novel Ca(2+)-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties.

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.

Amino Acid Sequence↗

PubMed1993

Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.

We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E2 generated high titers of anti-E1 and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified E1-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo, Lancet, in press) and generated protective immunity in chimpanzees (Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes.

Animals↗

PubMed1993

Characterization of a breast cancer cell differentiation factor that specifically activates the HER4/p180erbB4 receptor.

We recently reported the molecular cloning of HER4/p180erbB4, a new member of the epidermal growth factor receptor family, as well as its activation by a partially purified ligand (Plowman, G. D., Culouscou, J.-M., Whitney, G. S., Green, J. M., Carlton, G. W., Foy, L., Neubauer, M. G., and Shoyab, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1746-1750). In this report we describe the purification to homogeneity of a 45-kDa protein (p45) that induces the differentiation of MDA-MB-453 human breast cancer cells and stimulates the tyrosine phosphorylation of p180erbB4, the HER4-encoded protein. Hydrophobic interaction, ion-exchange, heparin, and size exclusion chromatographies were used to purify this p180erbB4 activator to homogeneity. N-terminal amino acid sequencing suggests that p45 is related to heregulin, a recently reported ligand for p185erbB2. Binding and cross-linking experiments demonstrated that p45 specifically binds to cells expressing recombinant p180erbB4 and not cells expressing recombinant p185erbB2.

Amino Acid Sequence↗