| 1. |
- Agaton, C., et al.
(författare)
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Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues
- 2003
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 2, s. 405-
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Tidskriftsartikel (refereegranskat)abstract
- Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.
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| 2. |
- Andersson, C., et al.
(författare)
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Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation
- 2000
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 238:02-jan, s. 181-193
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Tidskriftsartikel (refereegranskat)abstract
- We have previously reported a strategy for production in Escherichia coli of recombinant immunogens fused to a hydrophobic tag to improve their capacity to associate with an adjuvant formulation [Andersson et al., J. Immunol. Methods 222 (1999) 171]. Here, we describe a further development of the previous strategy and present significant improvements. In the novel system, the target immunogen is produced with an N-terminal affinity tag suitable for affinity purification, and a C-terminal hydrophobic tag, which should enable association through hydrophobic interactions of the immunogen with an adjuvant system, here being immunostimulating complexes (iscoms). Two different hydrophobic tags were evaluated: (i) a tag denoted M, derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA), and (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus. Furthermore, two alternative affinity tags were evaluated; the serum albumin-binding protein ABP, derived from streptococcal protein G, and the divalent IgG-binding ZZ-domains derived from SpA. A malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in this study. Four different fusion proteins, ABP-MS-M, ABP-MS-MI, ZZ-MS-M and ZZ-MS-MI, were thus produced, affinity purified and evaluated in iscom-incorporation experiments. All of the fusion proteins were found in the iscom fractions in analytical ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. In addition, these iscom preparations were demonstrated to induce MS-specific antibody responses upon immunisation of mice, confirming the successful incorporation into iscoms. The novel system for hydrophobic tagging of immunogens, with optional affinity and hydrophobic tags, gave expression levels that were increased ten to fifty-fold, as compared to the earlier reported system. We believe that the presented strategy would be a convenient way to achieve efficient adjuvant association for recombinant immunogens.
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| 3. |
- Andersson, Christin, et al.
(författare)
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In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation
- 2001
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Ingår i: Journal of Immunological Methods. - 0022-1759. ; 255:1-2, s. 135-148
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Tidskriftsartikel (refereegranskat)abstract
- We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment ΔSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-ΔSAG1 and His6-ABP-ΔSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-ΔSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, ΔSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that ΔSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.
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| 4. |
- Andersson, C., et al.
(författare)
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Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column
- 2001
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 34, s. 25-32
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Tidskriftsartikel (refereegranskat)abstract
- The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.
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| 5. |
- Andersson, C., et al.
(författare)
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Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunisation encoding a secreted RSV G protein-derived antigen
- 2000
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Ingår i: FEMS Immunology and Medical Microbiology. - 0928-8244 .- 1574-695X. ; 29:4, s. 247-253
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Tidskriftsartikel (refereegranskat)abstract
- Plasmid vectors encoding two different variants, one cytoplasmic and one secreted version, of a candidate vaccine BBG2Na to respiratory syncytial virus (RSV), were constructed and evaluated in a nucleic acid vaccination study. The two different vectors, which employed the Semliki Forest virus gene amplification system, were found to express BBG2Na appropriately in in vitro cell cultures. Immunisation of mice with the plasmid vectors elicited significant serum anti-BBG2Na IgG responses only in the mice receiving the plasmid encoding the secreted version of BBG2Na. Consistent with antibody induction data, sterilising lung protection against RSV-A challenge was also only observed in this group. These results indicate that the targeting of antigen expression (intracellular versus secreted) would be an important factor to consider in the design of nucleic acid vaccines.
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| 6. |
- Cano, F., et al.
(författare)
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Partial protection to respiratory syncytial virus (RSV) elicited in mice by intranasal immunization using live staphylococci with surface-displayed RSV-peptides
- 2000
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Ingår i: Vaccine. - 0264-410X .- 1873-2518. ; 18:24, s. 2743-2752
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Tidskriftsartikel (refereegranskat)abstract
- A live bacterial vaccine-delivery system based on the food-grade bacterium Staphylococcus carnosus was used for delivery of peptides from the G glycoprotein of human respiratory syncytial virus, subtype A (RSV-A). Three peptides, corresponding to the G protein amino acids, 144-159 (denoted G5), 190-203 (G9) and 171-188 (G4 S), the latter with four cysteine residues substituted for serines, were expressed by recombinant means as surface-exposed on three different bacteria, and their surface accessibility on the bacteria was verified by fluorescence-activated cell sorting (FACS). Intranasal immunization of mice with the live recombinant staphylococci elicited significant anti-peptide as well as anti-virus serum IgG responses of balanced IgG1/IgG2a isotype profiles, and upon viral challenge with 10(5) tissue culture infectious doses(50) (TCID50), lung protection was demonstrated for approximately half of the mice in the G9 and G4 S immunization groups. To our knowledge, this is the first study in which protective immunity to a viral pathogen has been evoked using food-grade bacteria as vaccine-delivery vehicles.
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| 7. |
- Carlsson, Jörgen, et al.
(författare)
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Polypeptides having binding affinity for HER2
- 2003
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Patent (populärvet., debatt m.m.)abstract
- A polypeptide is provided, which has a binding affinity for HER2 and which is related to a domain of staphylococcal protein A (SPA) in that the sequence of the polypeptide corresponds to the sequence of the SPA domain having from 1 to about 20 substitution mutations. Nucleic acid encoding the polypeptide, as well as expression vector and host cell for expressing the nucleic acid, are also provided. Also provided is the use of such a polypeptide as a medicament, and as a targeting agent for directing substances conjugated thereto to cells overexpressing HER2. Methods, and kits for performing the methods, are also provided, which methods and kits rely on the binding of the polypeptide to HER2.
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| 8. |
- Falk, Ronny, et al.
(författare)
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A high-stringency proteomics concept aimed for generation of antibodies specific for cDNAencoded proteins
- 2002
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 35:2, s. 75-82
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Tidskriftsartikel (refereegranskat)abstract
- A novel dual bacterial expression system, designed for high-throughput generation of antibodies specific for cDNA-encoded proteins, is presented. The concept involves parallel expression of cDNA-encoded proteins, in two vector systems, as fusions with two different tags, both enabling single-step affinity purification under denaturing conditions. One of the fusion tags includes a portion with documented immunopotentiating effect to stimulate antibody production, and the generated fusion proteins are used to elicit antibodies. The second fusion protein is used in an immobilized form as an affinity ligand to enrich, from the generated antisera, antibodies with selective reactivity to the cDNA-encoded part. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The five antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the five antibodies gave specific staining in Western-blot screening of various cell types and tissue homogenates. When the same five cDNAs were processed and analysed using a single-vector method, antibodies with a more non-specific staining were generated. We thus conclude that the presented dual-vector method offers a highly stringent strategy for generation of monospecific polyclonal antibodies.
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| 9. |
- Falk, Ronny, et al.
(författare)
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An improved dual-expression concept, generating high-quality antibodies for proteomics research
- 2003
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Ingår i: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 38, s. 231-239
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Tidskriftsartikel (refereegranskat)abstract
- A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.
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| 10. |
- Goetsch, L., et al.
(författare)
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Influence of administration dose and route on the immunogenicity and protective efficacy of BBG2Na, a recombinant respiratory syncytial virus subunit vaccine candidate
- 2000
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Ingår i: Vaccine. - 0264-410X .- 1873-2518. ; 18:24, s. 2735-2742
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Tidskriftsartikel (refereegranskat)abstract
- The immunogenicity and protective efficacy of BBG2Na, a novel recombinant respiratory syncytial virus subunit vaccine candidate, was assessed in BALB/c mice under various conditions of dose, administration route and number of immunisations. A single intra-peritoneal (i.p.) dose of 2 mu g, or two doses of 0.2 mu g, were sufficient to induce elevated RSV-A serum antibodies and sterilising lung protective immunity. Serum antibody titres were significantly boosted following second immunisations, but not a third. Of three routes of immunisation, i.p. induced the highest RSV-A antibody titres, followed in efficacy by the intramuscular (i.m.) and subcutaneous (s.c.) routes. Nonetheless, all three routes induced comparable and sterilising lung protection. In contrast, upper respiratory tract protection was observed only after i.p. vaccination, although significant viral titre reductions were evident following i.m. or s.c. immunisations. Interestingly, Pepscan analyses indicated that antibody epitope usage was highest in i.p. and lowest in i.m. immunised mice, respectively. Nonetheless, all routes resulted in antibody responses to known lung protective epitopes (protectopes). Thus, the prevention of serious lower respiratory tract disease, the principle goal of a RSV vaccine, but not URT infection, is dose dependent but unlikely to be influenced by the route of BBG2Na administration.
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| 11. |
- Graslund, S., et al.
(författare)
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Single-vector three-frame expression systems for affinity-tagged proteins
- 2002
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 215:1, s. 139-147
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Tidskriftsartikel (refereegranskat)abstract
- An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.
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| 12. |
- Gräslund, Susanne, et al.
(författare)
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A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products
- 2002
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 99:1, s. 41-50
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Tidskriftsartikel (refereegranskat)abstract
- An expression vector system has been developed, taking advantage of a novel, Staphylococcus aureus protein A (SPA)-binding affinity tag ZSPA-1, enabling straightforward affinity blotting procedures and efficient recovery by affinity purification of expressed gene products on readily available reagents and chromatography media. The 58 amino acid SPA-binding affinity tag ZSPA-1, was previously selected from a library constructed by combinatorial mutagenesis of a protein domain from SPA. An Escherichia coli expression vector for intracellular T7 promoter (PT7) driven production was constructed with an N-terminal dual affinity tag, consisting of a hexahistidyl (His6) tag in frame with the ZSPA-1 tag, thus allowing alternative affinity recovery methods. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and two alternative blotting procedures were developed for convenient screening of expression efficiencies. The five produced fusion proteins were recovered on both immobilized metal-ion affinity chromatography (IMAC) columns and on Protein A-based chromatography media, to allow comparative studies. It was found that the Protein A-based recovery resulted in the highest degree of purity, and furthermore, gene products that were produced as inclusion bodies could after denaturation be efficiently affinity purified on Protein A-Sepharose in the presence of 0.5 M guanidine hydrochloride. The convenience and robustness of the presented expression system should make it highly suitable for various high-throughput protein expression efforts.
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| 13. |
- Gulich, S., et al.
(författare)
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Engineering streptococcal protein G for increased alkaline stability
- 2002
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Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139 .- 1460-213X. ; 15:10, s. 835-842
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Tidskriftsartikel (refereegranskat)abstract
- Most protein-based affinity chromatography media are very sensitive towards alkaline treatment, which is a preferred method for regeneration and removal of contaminants from the purification devices in industrial applications. In a previous study, we concluded that a simple and straightforward strategy consisting of replacing asparagine residues could improve the stability towards alkaline conditions. In this study, we have shown the potential of this rationale by stabilizing an IgG-binding domain of streptococcal protein G, i.e. the C2 domain. In order to analyze the contribution of the different amino acids to the alkaline sensitivity of the domain we used a single point mutation strategy. Amino acids known to be susceptible towards high pH, asparagine and glutamine, were substituted for less-alkali-susceptible residues. In addition, aspartic acid residues were mutated to evaluate if the stability could be further increased. The stability of the different C2 variants was subsequently analyzed by exposing them to NaOH. The obtained results reveal that the most sensitive amino acid towards alkaline conditions in the structure of C2 is Asn36. The double mutant, C2(N7,36A), was found to be the most stable mutant constructed. In addition to the increased alkaline stability and also very important for potential use as an affinity ligand, this mutated variant also retains the secondary structure, as well as the affinity to the Fc fragment of IgG.
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| 14. |
- Hansson, M., et al.
(författare)
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Design and production of recombinant subunit vaccines
- 2000
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 32, s. 95-107
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Forskningsöversikt (refereegranskat)abstract
- The development of subunit vaccines is presently the main strategy being evaluated for prevention of infectious diseases. The use of recombinant-DNA techniques has facilitated the development of new principles for design and production of subunit vaccines. First of all, the properties of a target protein immunogen can be improved by the use of gene-fusion technology or by the creation of specific changes, to generate 'second-generation protein vaccines', Properties that can be modified include protein solubility, protein stability, in vivo half-lives, etc. In addition, for subunit protein vaccine candidates, the immunogenic properties can be significantly augmented by the addition of immunopotentiating tags or by means of targeting to immunoreactive sites. The recombinant subunit vaccine can furthermore be adapted by gene-fusion technology, to be efficiently incorporated into immunopotentiating adjuvant systems. Also in passive vaccination strategies, i.e. the use of antibodies or antibody fragments for prevention of infectious diseases, the recombinant strategies have become increasingly important. Humanized antibodies and antibody fusion. proteins represent common present anti-infectious-disease agents. The selected examples will indicate that recombinant strategies will indeed have an impact on the design, selection and production of recombinant proteins to be used in the prevention of infectious diseases.
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| 15. |
- Hansson, M., et al.
(författare)
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General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae
- 2002
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 210:2, s. 263-270
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Tidskriftsartikel (refereegranskat)abstract
- General expression vectors, designed for intracellular expression or secretion of recombinant proteins in the non-pathogenic Staphylococcus carnosus, were constructed. Both vector systems encode two different affinity tags, an upstream albumin binding protein and a downstream hexahistidyl peptide, and are furnished with cleavage sites for two site-specific proteases for optional affinity tag removal. To evaluate the novel vectors, the gene encoding the outer membrane protein A (OmpA) of Klebsiella pneumoniae was introduced into the vectors. Efficient production was demonstrated in both systems, although, as expected for OmpA fusions, somewhat better intracellularly, and the fusion proteins could be recovered as full-length products by affinity chromatography.
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| 16. |
- Hansson, M., et al.
(författare)
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Surface display on gram positive bacteria
- 2001
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Ingår i: Combinatorial chemistry & high throughput screening. - : Bentham Science Publishers Ltd.. - 1386-2073 .- 1875-5402. ; 4:2, s. 171-184
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Tidskriftsartikel (refereegranskat)abstract
- Heterologous surface display on Gram-positive bacteria was first described almost a decade ago and has since then developed into an active research area. Gram-positive bacterial surface display has today found a range of applications, in immunology, microbiology and biotechnology. Live bacterial vaccine delivery vehicles are being developed through the surface display of selected foreign antigens on the bacterial surfaces. In this field, second generation vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered Gram-positive bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other variants of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. This article explains the basis of Grampositive bacterial surface display, and discusses current uses and possible future trends of this emerging technology.
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| 17. |
- Jonasson, P., et al.
(författare)
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Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli
- 2002
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 35, s. 91-105
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Forskningsöversikt (refereegranskat)abstract
- Genetic strategies have been used for more than two decades to improve bacterial bioprocesses and to simplify recovery procedures. Such strategies include the design of efficient expression vectors and the improvement of bacterial production strains in different ways, e.g. by deletion of protease genes or engineering for overexpression of rare-codon tRNAs, foldases or chaperones. Gene multimerization is another such principle that has proved beneficial to improve production yields. Genetic strategies have furthermore been exploited to facilitate recovery processes by adapting the product for a particular purification principle. In this area, affinity fusions have been commonly used, but other principles, such as modified isoelectric point (pI) or hydrophobic properties have also been successfully investigated. A recent drastic step forward in the use of gene technology to improve recovery processes for recombinant proteins is the introduction of combinatorial protein engineering to generate tailor-made product-specific affinity ligands. This strategy, which allows efficient recovery of a recombinant protein in its native form, is likely to be increasingly used also in industrial-scale bioprocesses, since novel protein ligands have been described that can be sanitized using common industrial cleaning-in-p lace procedures. The examples presented in this review make it evident that genetic strategies will be of utmost importance in the future for facilitating production and recovery of recombinant proteins.
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| 18. |
- Jonasson, P., et al.
(författare)
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Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein
- 2000
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 76:03-feb, s. 215-226
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Tidskriftsartikel (refereegranskat)abstract
- An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.
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| 19. |
- Jorgensen, P. M., et al.
(författare)
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Characterisation of the human APC1, the largest subunit of the anaphase-promoting complex
- 2001
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 262:02-jan, s. 51-59
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Tidskriftsartikel (refereegranskat)abstract
- Accurate segregation of sister chromatids during mitosis is necessary to avoid the aneuploidy found in many cancers. The spindle checkpoint, which monitors the metaphase to anaphase transition, has been shown to be defective in cancers with chromosomal instability. This checkpoint regulates the anaphase-promoting complex or cyclosome (APC/C), a cell cycle ubiquitin ligase regulating among other things sister chromatid separation. We have previously investigated the mouse Apc1 protein (previously also called Tsg24), the largest subunit of the APC/C. We have now sequenced a full-length human APC1 cDNA, mapped its chromosomal location, and analysed its intron-exon boundaries. We have also investigated the RNA and protein expression of the Apc1 and other APC/C components in normal and cancer cells and the relative occurrence of expressed sequence tugs (ESTs) representing APC subunits from different tissues. The different APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they perform their functions as part of a complex. A difference from this pattern is however seen for the APC6, which in some cases is more strongly expressed, suggesting a special function for this protein in certain tissues and cell types.
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| 20. |
- Larsson, M., et al.
(författare)
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Expression profile viewer (ExProView) : A software tool for transcriptome analysis
- 2000
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Ingår i: Genomics. - : Elsevier BV. - 0888-7543 .- 1089-8646. ; 63:3, s. 341-353
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Tidskriftsartikel (refereegranskat)abstract
- A software tool, Expression Profile Viewer (ExProView), for analysis of gene expression profiles derived from expressed sequence tags (ESTs) and SAGE (serial analysis of gene expression) is presented. The software visualizes a complete set of classified transcript data in a two-dimensional array of dots, a virtual chip, in which each dot represents a known gene as characterized in the transcript databases Expressed Gene Anatomy Database or UniGene. The virtual chip display can be changed between representations of different conceptual systems for gene/protein classification and grouping. Four alternative projections are currently available: (i) cellular role, (ii) subcellular compartment, (iii) chromosome localization, and (iv) total UniGene display. However, the chip can be adapted to any other desired layout. By selecting dots, further information about the represented genes is obtained from the local database and WWW links. The software thus provides a visualization of global mRNA expression at the descriptive level and guides in the exploration of patterns of functional expression, while maintaining direct access to detailed information on each individual gene. To evaluate the software, public EST and SAGE gene expression data obtained from the Cancer Genome Anatomy Project at the National Center for Biotechnology Information were analyzed and visualized. A demonstration of the software is available at http://www.biochem.kth. se/exproview/.
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| 21. |
- Larsson, M., et al.
(författare)
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High-throughput protein expression of cDNA products as a tool in functional genomics
- 2000
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 80:2, s. 143-157
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Tidskriftsartikel (refereegranskat)abstract
- A proteomics approach has been developed aimed to allow high throughput analysis of protein products expressed from cDNA fragments (expressed sequence tags, ESTs). The concept relies on expression of gene products to generate specific antibodies for protein analysis, such as immunolocalization of the proteins on cellular and subcellular level. To evaluate the system, 55 cDNA clones with predominantly unknown function were selected from a mouse testis cDNA-library. A bacterial expression system was designed that allowed robust expression and easy purification. Protein levels between 15 and 80 mg l(-1) were obtained for 49 of the clones. Five clones were selected for immunization and all yielded functional antibodies that gave specific staining in Western blot screening of samples from various cell types. Furthermore, extensive immunolocalization information on subcellular level was obtained for three of the five clones. All generated data were stored in a relational database, and are made available through a web-interface (http://www.biochem.kth.se/multiscale/), which also provides relevant links and allows homology searches from the original sequences. The possibility to allow analysis of gene products from whole genomes using this 'localization proteomics' approach is discussed.
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| 22. |
- Larsson, M., et al.
(författare)
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The spatial and temporal expression of Tekt1, a mouse tektin C homologue, during spermatogenesis suggest that it is involved in the development of the sperm tail basal body and axoneme
- 2000
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Ingår i: European Journal of Cell Biology. - : Elsevier BV. - 0171-9335 .- 1618-1298. ; 79:10, s. 718-725
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Tidskriftsartikel (refereegranskat)abstract
- Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. Recently we described the sequence of the first mammalian tektin protein, Tekt1 (from mouse testis), which is most homologous with sea urchin tektin C. We have now investigated the temporal and spatial expression of Tekt1 during mouse male germ cell development. By in situ hybridization analysis TEKT1 RNA expression is detected in spermatocytes and in round spermatids in the mouse testis, Immunofluorescence microscopy analysis with anti-Tekt1 antibodies showed no distinct labeling of any subcellular structure in spermatocytes, whereas in round spermatids anti-Tekt1 antibodies co-localize with anti-ANA antibodies to the centrosome. At a later stage, elongating spermatids display a larger area of anti-Tekt1 staining at their caudal ends; as spermiogenesis proceeds, the anti-Tekt1 staining disappears. Together with other evidence, these results provide the first intraspecies evidence that Tekt1 is transiently associated with the centrosome, and indicates that Tekt1 is one of several tektins to participate in the nucleation of the flagellar axoneme of mature spermatozoa, perhaps being required to assemble the basal body.
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| 23. |
- Lehtio, J., et al.
(författare)
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Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain
- 2001
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Ingår i: FEMS Microbiology Letters. - 0378-1097 .- 1574-6968. ; 195:2, s. 197-204
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Tidskriftsartikel (refereegranskat)abstract
- The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells. since full-length proteins could be extracted and affinity-purified. Furthermore. surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization Tor the generation of whole-cell microbial tools Fur different applications will be discussed.
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| 24. |
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| 25. |
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| 26. |
- Ronnmark, J., et al.
(författare)
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Construction and characterization of affibody-Fc chimeras produced in Escherichia coli
- 2002
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Ingår i: JIM - Journal of Immunological Methods. - 0022-1759 .- 1872-7905. ; 261:02-jan, s. 199-211
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Tidskriftsartikel (refereegranskat)abstract
- Affibody-Fc chimeras were constructed by genetic fusion between different affibody affinity proteins with prescribed specificities and an Fc fragment derived from human I-G. Using affibody ligands previously selected for binding to respiratory syncytial virus (RSV) surface protein G and Thermus aquaticus (Taq) DNA polymerase, respectively, affibody-Fc fusion proteins showing spontaneous Fc fragment-mediated homodimerization via disulfide bridges were produced in Escherichia coli and affinity purified on protein A Sepharose from bacterial periplasms at yields ranging between 1 and 6 mg/l culture, Further characterization of the chimeras using biosensor technology showed that the affibody moieties have retained high selectivities for their respective targets after fusion to the Fc fragment. Avidity effects in the target binding were observed for the affibody-Fc chimeras compared to monovalent affibody fusion proteins, indicating that both affibody moieties in the chimeras were accessible and contributed in the binding. Fusion of a head-to-tail dimeric affibody moiety to the Fc fragment resulted in tetravalent affibody constructs which showed even more pronounced avidity effects. In addition, the Fc moiety of the chimeras was demonstrated to be specifically recognized by anti-human IgG antibody enzyme conjugates. One application for this class of artificial antibodies was demonstrated in a western blotting experiment in which one of the anti-RSV surface protein G affibody-Fc chimeras was demonstrated to be useful for specific detection of the target protein in a complex background consisting of a total E. coli lysate. The results show that through the replacement of the Fab portion of an antibody for an alternative binding domain based on a less complicated structure, chimeric proteins compatible with bacterial production routes containing both antigen recognition domains and Fc domains can be constructed. Such artificial antibodies should be interesting alternatives to, for example, whole antibodies or scFv-Fc fusions as detection devices and in diagnostic or therapeutic applications.
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| 27. |
- Samuelson, Patrik, et al.
(författare)
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Display of proteins on bacteria
- 2002
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 96:2, s. 129-154
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Forskningsöversikt (refereegranskat)abstract
- Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and disc-asses current uses and possible future trends of these emerging technologies.
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| 28. |
- Samuelson, Patrik, et al.
(författare)
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Staphylococcal surface display of metal-binding polyhistidyl peptides
- 2000
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 66:3, s. 1243-1248
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni2+- and Cd2+-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.
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| 29. |
- Ståhl, Stefan, et al.
(författare)
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Staphylococcal surface display and its applications
- 2000
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Ingår i: International Journal of Medical Microbiology. - : Elsevier BV. - 1438-4221 .- 1618-0607. ; 290:7, s. 571-577
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Forskningsöversikt (refereegranskat)abstract
- Novel surface proteins can be introduced onto the bacterial cell surface by recombinant means. Here, we describe the development of such display systems for two food-grade bacteria, Staphylococcus carnosus and Staphylococcus xylosus, and present how such engineered bacteria can be used in different applications. A study will be described in which such staphylococci were employed as vaccine delivery vehicles to elicit protective antibody responses to respiratory syncytial virus (RSV). The use of surface-engineered staphylococci as novel microbial biocatalysts, as a new type of whole-cell diagnostic devices or for adsorption of metal ions with potential environmental or biosensor applications, will also be discussed.
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| 30. |
- Wernérus, Henrik, et al.
(författare)
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Biotechnological applications for surface-engineered bacteria
- 2004
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 40, s. 209-228
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Forskningsöversikt (refereegranskat)abstract
- Display of heterologous proteins on the surface of micro-organisms, enabled by means of recombinant DNA technology, has become an increasingly popular strategy in microbiology, biotechnology and vaccinology. Both Gram-negative and Gram-positive bacteria have been investigated for potential applications. The present review will describe the most commonly used systems for bacterial display, with a focus on the biotechnology applications. Live bacterial vaccine-delivery vehicles have long been investigated through the surface display of foreign antigens and, recently, 'second-generation' vaccine-delivery vehicles have been generated by the addition of mucosal targeting signals, as a means to increase immune responses. Engineered bacteria have also the potential to act as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. They provide the potential for new types of whole-cell diagnostic devices, since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created, and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being explored. Certain bacteria have also been employed to display various polypeptide libraries for use as devices in in vitro selection applications. Part of the present review has been devoted to a more in-depth description of a promising Gram-positive display system, i.e. Staphylococcus carnosus, and its applications. The review describes the basic principles of the different bacterial display systems and discusses current uses and possible future trends of these emerging technologies.
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| 31. |
- Wernérus, Henrik, et al.
(författare)
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Engineering of staphylococcal surfaces for biotechnological applications
- 2002
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 96:1, s. 67-78
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Forskningsöversikt (refereegranskat)abstract
- Novel surface proteins can be introduced onto bacterial cell surfaces by recombinant means. Here, we describe various applications of two such display systems for the food-grade bacteria Staphylococcus carnosus and Staphylococcus xylosus, respectively. The achievements in the use of such staphylococci as live bacterial vaccine delivery vehicles will be described. Co-display of proteins and peptides with adhesive properties to enable targeting of the bacteria, have significantly improved the vaccine delivery potential. Recently, protective immunity to respiratory syncytial virus (RSV) could be evoked in mice by intranasal immunization using such 'second generation' vaccine delivery systems. Furthermore, antibody fragments and other 'affinity proteins' with capacity to specifically bind a certain protein, e.g. Staphylococcus aureus protein A-based affibodies, have been surface-displayed on staphylococci as initial efforts to create whole-cell diagnostic devices. Surface display of metal-binding peptides, or protein domains into which metal binding properties has been engineered by combinatorial protein engineering, have been exploited to create staphylococcal bioadsorbents for potential environmental or biosensor applications. The use of these staphylococcal surface display systems as alternatives for display of large protein libraries and subsequent affinity selection of relevant binding proteins by fluorescence-activated cell sorting (FACS) will be discussed.
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| 32. |
- Wernérus, Henrik, et al.
(författare)
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Fluorescence-activated cell sorting of specific affibody-displaying staphylococci
- 2003
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 69:9, s. 5328-5335
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Tidskriftsartikel (refereegranskat)abstract
- Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.
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| 33. |
- Wernérus, Henrik, et al.
(författare)
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Generation of metal-binding staphylococci through surface display of combinatorially engineered cellulose-binding domains
- 2001
-
Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 67:10, s. 4678-4684
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Tidskriftsartikel (refereegranskat)abstract
- Ni2+-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase CeI7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni2+-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni2+-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.
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| 34. |
- Wernérus, Henrik, et al.
(författare)
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Vector engineering to improve a staphylococcal surface display system
- 2002
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Ingår i: FEMS Microbiology Letters. - 0378-1097 .- 1574-6968. ; 212:1, s. 47-54
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Tidskriftsartikel (refereegranskat)abstract
- A previously developed expression system for surface display of heterologous proteins on the surface of Staphylococcus carnosus employs the secretion signals from a Staphylococcus hyicus lipase and the cell wall anchoring part of Staphylococcus aureus protein A (SpA) to achieve surface display of expressed recombinant proteins. The system has been successfully used in various applications but the vector has not been considered genetically stable enough to allow protein library display applications, which would be of obvious interest. A new set of vectors, differing in size and devoid of a phage f1 origin of replication, were constructed and evaluated in terms of bacterial growth characteristics and vector stability. Furthermore, surface expression of a model surface protein was monitored by an enzymatic whole-cell assay and flow cytometry. The engineered expression vectors demonstrated dramatically improved stability and growth properties and two of the novel vectors demonstrated retained high surface density of the displayed model protein. The flow cytometry was found to be a powerful toot for observing the surface density of displayed heterologous proteins, and would thus be a rational strategy for monitoring the optimisation of any surface display system. The implications of these improved display vectors for future protein library applications are discussed.
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| 35. |
- Wikman, Maria, et al.
(författare)
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Selection and characterization of HER2/neu-binding affibody ligands
- 2004
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 17:5, s. 455-462
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Tidskriftsartikel (refereegranskat)abstract
- Affibody (affibody) ligands that are specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) have been selected by phage display technology from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. The predominant variants from the phage selection were produced in Escherichia coli, purified by affinity chromatography, and characterized by biosensor analyses. Two affibody variants were shown to selectively bind to the extracellular domain of HER2/neu (HER2-ECD), but not to control proteins. One of the variants, denoted His6-ZHER2/neu:4, was demonstrated to bind with nanomolar affinity (approximately 50 nM) to the HER2-ECD molecule at a different site than the monoclonal antibody trastuzumab. Furthermore, radiolabeled His6-ZHER2/neu:4 affibody showed specific binding to native HER2/neu, overexpressed on the SKBR-3 tumor cell line. Such affibody ligands might be considered in tumor targeting applications for radionuclide diagnostics and therapy of adenocarcinomas such as breast and ovarian cancers.
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