| 1. |
- Aniander, Gustav, et al.
(author)
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Enhanced titer and quality of IgGs through alterations at the translation initiation sequence
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Other publication (other academic/artistic)abstract
- Enhancing recombinant expression in mammalian cells remains an important and valuable goal. Much work has been done to develop cell lines, growth conditions, and expression vectors. However, some parts of the expression vector have been comparatively neglected in this quest for improvement. One such part is the translation initiation site (TIS) sequence, which drives translation initiation by increasing ribosomal recognition of the start codon. Using earlier work on what nucleotides could be exchanged for increased expression, we here present a novel TIS sequence. This TIS sequence increased titer in transient settings 4-fold while yielding 13-33 % increase in titer in final selected stable clones. This increase also comes with an increase in quality through reduction in non-paired chains, shift in charged species distribution, whilst maintaining the glycosylation profile. This increase in both titer and quality in transient and stable settings showing that TIS sequence engineering is of great interest for optimizing recombinant production in mammalian cells.
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| 2. |
- Cano, F., et al.
(author)
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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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In: Vaccine. - 0264-410X .- 1873-2518. ; 18:24, s. 2743-2752
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Journal article (peer-reviewed)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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| 3. |
- Gustavsson, Martin, et al.
(author)
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Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli
- 2015
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14:1
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Journal article (peer-reviewed)abstract
- Background: Salmonella enterica serovar Enteritidis (SE) is one of the most potent pathogenic Salmonella serotypes causing food-borne diseases in humans. We have previously reported the use of the β-autotransporter AIDA-I to express the Salmonella flagellar protein H:gm and the SE serotype-specific fimbrial protein SefA at the surface of E. coli as live bacterial vaccine vehicles. While SefA was successfully displayed at the cell surface, virtually no full-length H:gm was exposed to the medium due to extensive proteolytic cleavage of the N-terminal region. In the present study, we addressed this issue by expressing a truncated H:gm variant (H:gmd) covering only the serotype-specific central region. This protein was also expressed in fusion to SefA (H:gmdSefA) to understand if the excellent translocation properties of SefA could be used to enhance the secretion and immunogenicity. Results: H:gmd and H:gmdSefA were both successfully translocated to the E. coli outer membrane as full-length proteins using the AIDA-I system. Whole-cell flow cytometric analysis confirmed that both antigens were displayed and accessible from the extracellular environment. In contrast to H:gm, the H:gmd protein was not only expressed as full-length protein, but it also seemed to promote the display of the protein fusion H:gmdSefA. Moreover, the epitopes appeared to be recognized by HT-29 intestinal cells, as measured by induction of the pro-inflammatory interleukin 8. Conclusions: We believe this study to be an important step towards a live bacterial vaccine against Salmonella due to the central role of the flagellar antigen H:gm and SefA in Salmonella infections and the corresponding immune responses against Salmonella.
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| 4. |
- Hansson, M., et al.
(author)
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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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In: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 210:2, s. 263-270
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Journal article (peer-reviewed)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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| 5. |
- Hansson, M., et al.
(author)
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Surface display on gram positive bacteria
- 2001
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In: Combinatorial chemistry & high throughput screening. - : Bentham Science Publishers Ltd.. - 1386-2073 .- 1875-5402. ; 4:2, s. 171-184
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Journal article (peer-reviewed)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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| 6. |
- Jarmander, Johan, 1983-, et al.
(author)
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A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli
- 2012
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 11
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Journal article (peer-reviewed)abstract
- Background: The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H: gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results: The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His(6)-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H: gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His(6)-tag facing the medium. Conclusions: Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His(6)-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.
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| 7. |
- Johansson, Jonas, et al.
(author)
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The structure of <1 1 1 > B oriented GaP nanowires
- 2007
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In: Journal of Crystal Growth. - : Elsevier BV. - 0022-0248. ; 298, s. 635-639
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Journal article (peer-reviewed)abstract
- Nanowires of zinc blende crystal structure, grown in the < 111 > B direction usually have a large number of twin plane defects. In order to investigate this phenomenon, we grow GaP nanowires with metal-organic vapor phase epitaxy. By rotating the nanowires in a high resolution transmission electron microscope, we show that the nanowire segments between the twin planes are of octahedral shape and are terminated by {111} facets. Due to the alternating orientations of these twin octahedra, the sidewalls of the nanowires can be described as microfaceted surfaces with an overall orientation of 11 1 2}, but composed of alternating 11 1 I}A and {1 I 1}B facets. Moreover, the segment thicknesses follow exponential distributions, which show that there is a certain probability of twin plane formation, which is independent of segment thickness. (c) 2006 Elsevier B.V. All rights reserved.
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| 8. |
- Kostallas, George, et al.
(author)
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Construction, expression and characterization of TEV protease mutants engineered for improved solubility
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Other publication (other academic/artistic)abstract
- In recent years, the highly sequence specific tobacco etch virus protease (TEVp) has emerged as one of the most popular and widely used reagents for removal of fusion tags from target proteins. Its use, however, has been hampered due to relatively poor solubility and inefficient expression in E. coli. Although a lot of progress has been made, there is still need for new and improved TEVp variants. Recently, two different gain-of-function TEVp mutants were described; one containing the substitutions L56V/S135G, which conferred improved solubility and activity in vitro, while the other mutant, containing the substitutions T17S/N68D/I77V, was claimed to yield more soluble protease than the wild-type (wt) protease upon overexpression in E. coli. Here, we analyzed if the L56V/S135G substitutions could promote increased solubility also in vivo, as that would be beneficial to TEVp production and had never been investigated before. We also intended to create a novel, and hopefully superior, TEVp variant with all five mutations combined (T17S/L56V/N68D/S135G/I77V) in a single protease molecule. This variant and the two parental TEVp variants as well as the wt protease, were all expressed in E. coli and characterized with respect to the expression levels, solubility and activity using several different techniques; among them, a newly developed fluorescence-assisted whole-cell assay that directly reports on the apparent protease activity in vivo. Our results show that the L56V/S135G substitutions improve the solubility not only in vitro but also in vivo, which did hold true for the activity as well. Disappointingly, the protease variant containing all five substitutions (T17S/L56V/N68D/S135G/I77V) did not show the best performance, which instead the L56V/S135G variant did. In contrast to an earlier report, we show that the substitutions T17S/N68D/I77V, did not improve the TEVp solubility. In fact, they reduced the activity, and even appeared to have a slightly negative effect on solubility, of all protease constructs in which they were present. Thus, the best current and most promising TEVp variant for future protease engineering efforts, towards improved expression properties and enhanced catalytic efficiency, are those containing the L56V/S135G substitutions.
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| 9. |
- Kostallas, George
(author)
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Intracellular systems for characterization and engineering of proteases and their substrates
- 2011
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Doctoral thesis (other academic/artistic)abstract
- Over the years, the view on proteases as relatively non-specific protein degradation enzymes, mainly involved in food digestion and intracellular protein turnover, has shifted and they are now recognized as key regulators of many biological processes that determine the fate of a cell. Besides their biological role, proteases have emerged as important tools in various biotechnical, industrial and medical applications. At present, there are worldwide efforts made that aim at deciphering the biological role of proteases and understanding their mechanism of action in greater detail. In addition, with the growing demand of novel protease variants adapted to specific applications, protease engineering is attracting a lot of attention. With the vision of contributing to the field of protein science, we have developed a platform for the identification of site-specific proteolysis, consisting of two intracellular genetic assays; one fluorescence-based (Paper I) and one antibiotic resistance-based (Paper IV). More specifically, the assays take advantage of genetically encoded short-lived reporter substrates that upon cleavage by a coexpressed protease confer either increased whole-cell fluorescence or antibiotic resistance to the cells in proportion to the efficiency with which the substrates are processed. Thus, the fluorescence-based assay is highly suitable for high-throughput analysis of substrate processing efficiency by flow cytometry analysis and cell sorting, while the antibiotic resistance assay can be used to monitor and identify proteolysis through (competitive) growth in selective media. By using the highly sequence specific tobacco etch virus protease (TEVp) as a model in our systems, we could show that both allowed for (i) discrimination among closely related substrate peptides (Paper I & IV) and (ii) enrichment and identification of the best performing substrate-protease combination from a background of suboptimal variants (Paper I & IV). In addition, the fluorescence-based assay was used successfully to determine the substrate specificity of TEVp by flow cytometric screening of large combinatorial substrate libraries (Paper II), and in a separate study also used as one of several methods for the characterization of different TEVp mutants engineered for improved solubility (Paper III). We believe that our assays present a new and promising path forward for high-throughput substrate profiling of proteases, directed evolution of proteases and identification of protease inhibitors, which all are areas of great biological, biotechnical and medical interest.
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| 10. |
- Kostallas, George, et al.
(author)
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Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates
- 2010
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In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 76:22, s. 7500-7508
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Journal article (peer-reviewed)abstract
- We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1: 100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors.
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