| 1. |
- Jansson, Ronnie, et al.
(författare)
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Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris
- 2016
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 11:5, s. 687-699
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Tidskriftsartikel (refereegranskat)abstract
- Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.
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| 2. |
- Rasool, Kashif, et al.
(författare)
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Comprehensive insights into sustainable conversion of agricultural and food waste into microbial protein for animal feed production
- 2023
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Ingår i: Reviews in Environmental Science and Biotechnology. - : Springer. - 1569-1705 .- 1572-9826. ; 22:2, s. 527-562
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Forskningsöversikt (refereegranskat)abstract
- The growing global population and higher living standards instantly demand the transition in the direction of a sustainable food system. A substantial section of means and agricultural lands are presently committed to protein-rich feed production to rear livestock for human consumption. Conversely, accelerated farming activities and the food industry have rendered a drastic increase in waste which impair the economic and environmental sustainability of the ecosystem. This situation emerges the need for developing an integrated technology for waste management and to improve sustainability footprints. Microbial protein (MP) production based on renewable electron and carbon sources has the potential as a substitute protein source. MP production for animal feed use is growing fast and is derived from bacteria, algae, and fungi including yeast. MP produced from all types of microbes is currently commercialized and in use. However, novel methods and processes are also under investigation to make MP production more economical and sustainable. Current research on MP has concentrated on the valorization of waste materials by using high protein content-containing microorganisms, which can then be used in animal feed. Using such kind of integrated approach, the agroindustry waste resources upcycling can contribute towards finding sustainable, cheaper, and environment-friendly protein sources. This review first describes the potential waste feedstock for MP production and summarizes the recent progress in the application of MP-producing microorganisms including fungus, yeast, bacteria, and phototrophic microbes. Bioprocesses, and production technology advances for MP production have been explored and discussed in detail. Finally, the MP application as animal feed, its challenges, and future perspectives in research have been evaluated.
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| 3. |
- Sepehri, Sobhan, 1986, et al.
(författare)
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Volume-amplified magnetic bioassay integrated with microfluidic sample handling and high-Tc SQUID magnetic readout
- 2018
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Ingår i: APL Bioengineering. - : AIP Publishing. - 2473-2877. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- A bioassay based on a high-Tc superconducting quantum interference device (SQUID) reading out functionalized magnetic nanoparticles (fMNPs) in a prototype microfluidic platform is presented. The target molecule recognition is based on volume amplification using padlock-probe-ligation followed by rolling circle amplification (RCA). The MNPs are functionalized with single-stranded oligonucleotides, which give a specific binding of the MNPs to the large RCA coil product, resulting in a large change in the amplitude of the imaginary part of the ac magnetic susceptibility. The RCA products from amplification of synthetic Vibrio cholera target DNA were investigated using our SQUID ac susceptibility system in microfluidic channel with an equivalent sample volume of 3 μl. From extrapolation of the linear dependence of the SQUID signal versus concentration of the RCA coils, it is found that the projected limit of detection for our system is about 1.0 e5 RCA coils (0.2e−18 mol), which is equivalent to 66 fM in the 3 μl sample volume. This ultra-high magnetic sensitivity and integration with microfluidic sample handling are critical steps towards magnetic bioassays for rapid detection of DNA and RNA targets at the point of care.
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| 4. |
- Bergenholtz, Sa Schoug, et al.
(författare)
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A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying
- 2012
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Ingår i: Cryobiology. - : Elsevier BV. - 0011-2240 .- 1090-2392. ; 64:3, s. 152-159
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Tidskriftsartikel (refereegranskat)abstract
- Freeze-drying of bacterial cells with retained viability and activity after storage requires appropriate formulation, i.e. mixing of physiologically adapted cell populations with suitable protective agents, and control of the freeze-drying process. Product manufacturing may alter the clinical effects of probiotics and it is essential to identify and understand possible factor co-dependencies during manufacturing. The physical solid-state behavior of the formulation and the freeze-drying parameters are critical for bacterial survival and thus process optimization is important, independent of strain. However, the maximum yield achievable is also strain-specific and strain survival is governed by e.g. medium, cell type, physiological state, excipients used, and process. The use of preferred compatible solutes for cross-protection of Lactobacilli during industrial manufacturing may be a natural step to introduce robustness, but knowledge is lacking on how compatible solutes, such as betaine, influence formulation properties and cell survival. This study characterized betaine formulations, with and without sucrose, and tested these with the model lactic acid bacteria Lactobacillus coryniformis Si3. Betaine alone did not act as a lyo-protectant and thus betaine import prior to freeze-drying should be avoided. Differences in protective agents were analyzed by calorimetry, which proved to be a suitable tool for evaluating the characteristics of the freeze-dried end products.
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| 5. |
- Jocic, Simonne, et al.
(författare)
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Fabrication of user-friendly and biomimetic 1,1′-carbonyldiimidazole cross-linked gelatin/agar microfluidic devices
- 2017
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Ingår i: Materials Science and Engineering C. - : Elsevier BV. - 0928-4931 .- 1873-0191. ; 76, s. 1175-1180
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a straightforward technique for fabricating user-friendly and biomimetic microfluidic devices out of a gelatin/agar gel cross-linked with 1,1′-carbonyldiimidazole. The fabrication procedure requires only inexpensive starting materials such as glass capillaries and wires to mold 3D cylindrical channels into the gel with the possibility of achieving channel diameters of 375 μm and 1000 μm. We demonstrate that the channel absent of gel injury can retain fluid within its dimensions for at least 7 h. We also show that the device material does not autofluoresce nor provide hindrances with fluorescent imaging. A discussion of the chemical linkage identities of cross-linked gelatin/agar is included via ATR-FTIR studies. Crosslinking of the gelatin/agar is further confirmed by the lack of a gel to sol transition at physiological temperature as assessed by DSC measurements. SEM micrographs that demonstrate the 100 nm mean pore width of the cross-linked gelatin/agar are provided. This device is considered biomimetic because it represents components present in the natural extracellular matrix such as collagen and proteoglycans in the form of cross-linked gelatin/agar.
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| 6. |
- Anderson, Henrik, 1975-
(författare)
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Development of Electroacoustic Sensors for Biomolecular Interaction Analysis
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biomolecular interaction analysis to determine the kinetics and affinity between interacting partners is important for the fundamental understanding of biology, as well as for the development of new pharmaceutical substances. A quartz crystal microbalance instrument suitable for kinetics and affinity analyses of interaction events was developed. The functionality of the sensor system was demonstrated by development of an assay for relative affinity determination of lectin-carbohydrate interactions.Sensor surfaces allowing for effective immobilization of one interacting partner is a key functionality of a biosensor. Here, three different surfaces and immobilization methods were studied. First, optimized preparation conditions for sensor surfaces based on carboxyl-terminated self assembled monolayers were developed and were demonstrated to provide highly functional biosensor surfaces with low non-specific binding. Second, a method allowing for immobilization of very acidic biomolecules based on the use of an electric field was developed and evaluated. The electric field made it possible to immobilize the highly acidic C-peptide on a carboxylated surface. Third, a method for antibody immobilization on a carboxyl surface was optimized and the influence of immobilization pH on the immobilization level and antigen binding capacity was thoroughly assessed. The method showed high reproducibility for a set of antibodies and allowed for antibody immobilization also at low pH.Three broadly different strategies to increase the sensitivity of electroacoustic sensors were explored. A QCM sensor with small resonator electrodes and reduced flow cell dimensions was demonstrated to improve the mass transport rate to the sensor surface. The use of polymers on QCM sensor surfaces to enhance the sensor response was shown to increase the response of an antibody-antigen model system more than ten-fold. Moreover, the application of high frequency thin film bulk acoustic resonators for biosensing was evaluated with respect to sensing range from the surface. The linear detection range of the thin film resonator was determined to be more than sufficient for biosensor applications involving, for instance, antibody-antigen interactions. Finally, a setup for combined frequency and resistance measurements was developed and was found to provide time resolved data suitable for kinetics determination.
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| 7. |
- Anderson, Henrik, et al.
(författare)
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Systematic investigation of biomolecular interactions using combined frequency and motional resistance measurements
- 2011
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Ingår i: Sensors and actuators. B, Chemical. - : Elsevier BV. - 0925-4005 .- 1873-3077. ; 153:1, s. 135-144
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Tidskriftsartikel (refereegranskat)abstract
- The resonance frequency of acoustic biosensors is today used as a label-free technique for detecting mass changes on sensor surfaces. In combination with an appropriate continuous flow system it has earlier been used for affinity and kinetic rate determination. Here, we assess the potential of a modified acoustic biosensor, monitoring also the real-time dissipation through the resistance of the sensor, to obtain additional kinetic information related to the structure and conformation of the molecules on the surface. Actual interaction studies, including an attempt to determine avidity, are presented along with thorough verification of the experimental setup utilizing true viscous load exposure together with protein and DNA immobilizations. True viscous loads show a linear relationship between resistance and frequency as expected. However, in the interaction studies between antibodies and proteins, as well as in the immobilization of DNA and proteins, higher surface concentrations of interacting molecules led to a decrease (i.e. deviation from the linear trend) in the differential resistance to frequency ratio. This is interpreted as increased surface rigidity at higher surface concentrations of immobilized molecules. Consequently, studies that aim at obtaining biological binding information, such as avidity, from real-time resistance and dissipation data should be conducted at low surface concentrations. In addition, the differential resistance to frequency relationship was found to be highly dependent on the rigidity of the preceding layer(s) of immobilized molecules. This dependence can be utilized to obtain a higher signal-to-noise ratio for resistance measurement by using low surface densities of immobilized interaction partners.
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| 8. |
- Caldwell, Karin D., et al.
(författare)
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The Origin of Sepahdex
- 2006
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Ingår i: GIT Laboratory Journal: Europe. ; 10:5, s. 18-20
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 9. |
- Carlesso, Antonio, 1990, et al.
(författare)
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Yeast as a tool for membrane protein production and structure determination
- 2022
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Ingår i: Fems Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 22:1
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Forskningsöversikt (refereegranskat)abstract
- Although the majority of eukaryotic MEMBRANE PROTEIN structures are DERIVED FROM PROTEINS produced in HEK293 and insect cells, the authors show here the importance of yeast as a production host and its role as an essential player in the production of eukaryotic membrane proteins for structural and functional analysis. Membrane proteins are challenging targets to functionally and structurally characterize. An enduring bottleneck in their study is the reliable production of sufficient yields of stable protein. Here, we evaluate all eukaryotic membrane protein production experiments that have supported the deposition of a high-resolution structure. We focused on the most common yeast host systems, Saccharomyces cerevisiae and Pichia pastoris. The first high-resolution structure of a membrane protein produced in yeast was described in 1999 and today there are 186 structures of alpha-helical membrane proteins, representing 101 unique proteins from 37 families. Homologous and heterologous production are equally common in S. cerevisiae, while heterologous production dominates in P. pastoris, especially of human proteins, which represent about one-third of the total. Investigating protein engineering approaches (78 proteins from seven families) demonstrated that the majority contained a polyhistidine tag for purification, typically at the C-terminus of the protein. Codon optimization and truncation of hydrophilic extensions were also common approaches to improve yields. We conclude that yeast remains a useful production host for the study of alpha-helical membrane proteins.
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| 10. |
- Cheng, Wing-Shing, 1974-
(författare)
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TARP Promoter-Based Prostate Cancer Gene Therapy : From Development to Application
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Prostate cancer is one leading cause of cancer-related death among men in Western countries. The standard therapies for localized prostate cancer include radical prostatectomy and radiation therapy. Such measures are relatively effective in the short term, but many patients ultimately relapse. These patients may benefit from a combination of standard therapy and oncolytic virus therapy. My work aimed to develop viruses for this purpose.TARP is a protein that in males is specifically expressed in prostate epithelial and cancer cells. In my thesis, I characterized the TARP promoter and showed that TARP expression is regulated at the transcriptional level by testosterone through binding of the androgen receptor in the proximal TARP promoter. I further developed TARP promoter-based regulatory sequences for prostate-specific gene expression. A sequence comprising a PSA enhancer, a PSMA enhancer and the TARP promoter was constructed and designated PPT. An adenoviral vector containing the PPT sequence shielded from transcriptional interference by an H19 insulator showed high prostate-specific transcriptional activity in human cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer the PPT sequence is not active. Therefore, a two-step transcriptional amplification (TSTA) system was used together with the PPT sequence to develop an adenovirus that confers prostate-specific transgene expression also in murine cells.I constructed a conditionally replicating adenovirus where the E1A gene expression is controlled by an H19 insulator-shielded PPT regulatory sequence, Ad[I/PPT-E1A]. This virus exhibited absolute prostate specificity in terms of E1A expression, viral replication and cytolysis in vitro and in vivo. Importantly, our virus is active both in the presence and absence of testosterone, which may prove beneficial for patients treated by hormonal withdrawal. Hopefully, my work will improve existing gene therapy strategies for prostate cancer and in the long term improve the prognosis for patients with prostate cancer.
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