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- Nyblom, Anna Maria, 1975, et al.
(author)
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Exceptional overproduction of a functional human membrane protein
- 2007
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In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 56:1, s. 110-120
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Journal article (peer-reviewed)abstract
- Eukaryotic-especially human-membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQPI in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. (c) 2007 Elsevier Inc. All rights reserved.
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| 2. |
- Carlander, A, et al.
(author)
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Clinical evaluation of a dual-side readout technique computed radiography system in chest radiography of premature neonates.
- 2008
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In: Acta radiologica (Stockholm, Sweden : 1987). - : SAGE Publications. - 1600-0455 .- 0284-1851. ; 49:4, s. 468-74
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Journal article (peer-reviewed)abstract
- BACKGROUND: Recently, the dual-side readout technique has been introduced in computed radiography, leading to an increase in detective quantum efficiency (DQE) compared with the single-side readout technique. PURPOSE: To evaluate if the increase in DQE with the dual-side readout technique results in a higher clinical image quality in chest radiography of premature neonates at no increase in radiation dose. MATERIAL AND METHODS: Twenty-four chest radiographs of premature neonates were collected from both a single-side readout technique system and a double-side readout technique system. The images were processed in the same image-processing station in order for the comparison to be only dependent on the difference in readout technique. Five radiologists rated the fulfillment of four image quality criteria, which were based on important anatomical landmarks. The given ratings were analyzed using visual grading characteristics (VGC) analysis. RESULTS: The VGC analysis showed that the reproduction of the carina with the main bronchi and the thoracic vertebrae behind the heart was better with the dual-side readout technique, whereas no significant difference for the reproduction of the central vessels or the peripheral vessels could be observed. CONCLUSIONS: The results indicate that the higher DQE of the dual-side readout technique leads to higher clinical image quality in chest radiography of premature neonates at no increase in radiation dose.
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| 7. |
- Malmberg, Emily, 1978, et al.
(author)
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Increased levels of mucins in the cystic fibrosis mouse small intestine, and modulator effects of the Muc1 mucin expression.
- 2006
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In: American journal of physiology. Gastrointestinal and liver physiology. - : American Physiological Society. - 0193-1857 .- 1522-1547. ; 291:2
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Journal article (peer-reviewed)abstract
- The mouse model (Cftr(tm1UNC)/Cftr(tm1UNC)) for cystic fibrosis (CF) shows mucus accumulation and increased Muc1 mucin mRNA levels due to altered splicing (Hinojosa-Kurtzberg AM, Johansson MEV, Madsen CS, Hansson GC, and Gendler SJ. Am J Physiol Gastrointest Liver Physiol 284: G853-G862, 2003). However, it is not known whether Muc1 is a major mucin contributing to the increased mucus and why CF/Muc1-/- mice show lower mucus accumulation. To address this, we have purified mucins from the small intestine of CF mice using guanidinium chloride extraction, ultracentrifugation, and gel filtration and analyzed them by slot blot, gel electrophoresis, proteomics, and immunoblotting. Normal and CF mice with wild-type (WT) Muc1 or Muc1-/- or that are transgenic for human MUC1 (MUC1.Tg, on a Muc1-/- background) were analyzed. The total amount of mucins, both soluble and insoluble in guanidinium chloride, increased up to 10-fold in the CF mice compared with non-CF animals, whereas the CF mice lacking Muc1 showed intermediate levels between the CF and non-CF mice. However, the levels of Muc3 (orthologue of human MUC17) were increased in the CF/Muc1-/- mice compared with the CF/MUC1.Tg animals. The amount of MUC1 mucin was increased several magnitudes in the CF mice, but MUC1 did still not appear to be a major mucin. The amount of insoluble mucus of the large intestine was also increased in the CF mice, an effect that was partially restored in the CF/Muc1-/- mice. The thickness of the firmly adherent mucus layer of colon in the Muc1-/- mice was significantly lower than that of WT mice. The results suggest that MUC1 is not a major component in the accumulated mucus of CF mice and that MUC1 can influence the amount of other mucins in a still unknown way.
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| 8. |
- Persson, Jonas, et al.
(author)
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Molecular evolution of specific human antibody against MUC1 mucin results in improved recognition of the antigen on tumor cells.
- 2009
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In: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. - : Springer Science and Business Media LLC. - 1423-0380 .- 1010-4283. ; 30:4, s. 221-31
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Journal article (peer-reviewed)abstract
- The MUC1 mucin is differentially expressed and glycosylated in cancer tissue as opposed to healthy tissue. Due to these differences, MUC1 is considered a potential biomarker suitable for cancer diagnosis and therapy. In a previous study, the human MUC1-specific antibody 12ESC-6 was able to bind a sequence variant of the tandem repeat of MUC1 that is not recognized by many other MUC1-specific antibodies. It was also found to bind efficiently to MUC1-carrying cells. We have now used 12ESC-6 as starting point for random mutagenesis to isolate variants with improved ability to bind MUC1 in human tumor tissue. The resulting 12ESC-6 variants were shown to recognize not only the naked MUC1 tandem repeat but even more so glycosylated variants thereof, in particular those carrying the GalNAc (Tn) glycoform. Selected variants of 12ESC-6 demonstrated improved staining of MUC1 on cell lines using flow cytometry and improved staining of the antigen in breast tumor tissue by immunohistochemistry. Molecular evolution and specific fine-tuning thus have the potential to improve the performance of antibody specificities targeting tumor-associated epitopes on MUC1 mucin.
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| 9. |
- Quiding-Järbrink, Marianne, 1964, et al.
(author)
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Decreased IgA antibody production in the stomach of gastric adenocarcinoma patients.
- 2009
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In: Clinical immunology. - : Elsevier BV. - 1521-7035 .- 1521-6616. ; 131:3, s. 463-71
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Journal article (peer-reviewed)abstract
- Gastric adenocarcinoma is closely associated with Helicobacter pylori infection. It is also much more frequent in patients with common variable immunodeficiency or selective IgA-deficiency than in the general population. To investigate a possible link between local antibody production and gastric tumors, we studied gastric B cell infiltration and local IgA production in patients with H. pylori induced gastric adenocarcinomas. These studies showed that total and H. pylori-specific IgA antibody levels were substantially lower in gastric tissue from the cancer patients compared to those from asymptomatic H. pylori carriers. However, serum IgA levels were similar in the cancer patients and asymptomatic carriers. As could be expected, H. pylori infected asymptomatic carriers had considerably increased IgA antibody levels compared to uninfected subjects. We conclude that patients suffering from gastric adenocarcinoma have a dramatically decreased local IgA production in the stomach compared to asymptomatic H. pylori infected individuals.
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