Publications by authors named "Schlapschy M"

24 Publications

  • Page 1 of 1

Hsp70 in Liquid Biopsies-A Tumor-Specific Biomarker for Detection and Response Monitoring in Cancer.

Cancers (Basel) 2021 Jul 23;13(15). Epub 2021 Jul 23.

Center for Translational Cancer Research (TranslaTUM), Radiation Immuno-Oncology Group, Technical University of Munich (TUM), Klinikum rechts der Isar, Einsteinstr. 25, 81675 Munich, Germany.

In contrast to normal cells, tumor cells of multiple entities overexpress the Heat shock protein 70 (Hsp70) not only in the cytosol, but also present it on their plasma membrane in a tumor-specific manner. Furthermore, membrane Hsp70-positive tumor cells actively release Hsp70 in small extracellular vesicles with biophysical characteristics of exosomes. Due to conformational changes of Hsp70 in a lipid environment, most commercially available antibodies fail to detect membrane-bound and vesicular Hsp70. To fill this gap and to assess the role of vesicular Hsp70 in circulation as a potential tumor biomarker, we established the novel complete (comp)Hsp70 sandwich ELISA, using two monoclonal antibodies (mAbs), that is able to recognize both free and lipid-associated Hsp70 on the cell surface of viable tumor cells and on small extracellular vesicles. The epitopes of the mAbs cmHsp70.1 (aa 451-461) and cmHsp70.2 (aa 614-623) that are conserved among different species reside in the substrate-binding domain of Hsp70 with measured affinities of 0.42 nM and 0.44 nM, respectively. Validation of the compHsp70 ELISA revealed a high intra- and inter-assay precision, linearity in a concentration range of 1.56 to 25 ng/mL, high recovery rates of spiked liposomal Hsp70 (>84%), comparable values between human serum and plasma samples and no interference by food intake or age of the donors. Hsp70 concentrations in the circulation of patients with glioblastoma, squamous cell or adeno non-small cell lung carcinoma (NSCLC) at diagnosis were significantly higher than those of healthy donors. Hsp70 concentrations dropped concomitantly with a decrease in viable tumor mass upon irradiation of patients with approximately 20 Gy (range 18-22.5 Gy) and after completion of radiotherapy (60-70 Gy). In summary, the compHsp70 ELISA presented herein provides a sensitive and reliable tool for measuring free and vesicular Hsp70 in liquid biopsies of tumor patients, levels of which can be used as a tumor-specific biomarker, for risk assessment (i.e., differentiation of grade III vs. IV adeno NSCLC) and monitoring of therapeutic outcomes.
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http://dx.doi.org/10.3390/cancers13153706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8345117PMC
July 2021

Molecular recognition of structurally disordered Pro/Ala-rich sequences (PAS) by antibodies involves an Ala residue at the hot spot of the epitope.

J Mol Biol 2021 09 20;433(18):167113. Epub 2021 Jun 20.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany; XL-protein GmbH, Lise-Meitner-Strasse 30, 85354 Freising, Germany. Electronic address:

Pro/Ala-rich sequences (PAS) are polypeptides that were developed as a biological alternative to poly-ethylene glycol (PEG) to generate biopharmaceuticals with extended plasma half-life. Like PEG, PAS polypeptides are conformationally disordered and show high solubility in water. Devoid of any charged or prominent hydrophobic side chains, these biosynthetic polymers represent an extreme case of intrinsically disordered proteins. Despite lack of immunogenicity of PAS tags in numerous animal studies we now succeeded in generating monoclonal antibodies (MAbs) against three different PAS versions. To this end, mice were immunized with a PAS#1, P/A#1 or APSA 40mer peptide conjugated to keyhole limpet hemocyanin as highly immunogenic carrier protein. In each case, one MAb with high binding activity and specificity towards a particular PAS motif was obtained. The apparent affinity was strongly dependent on the avidity effect and most pronounced for the bivalent MAb when interacting with a long PAS repeat. X-ray structural analysis of four representative anti-PAS Fab fragments in complex with their cognate PAS epitope peptides revealed interactions dominated by hydrogen bond networks involving the peptide backbone as well as multiple Van der Waals contacts arising from intimate shape complementarity. Surprisingly, Ala, the L-amino acid with the smallest side chain, emerged as a crucial feature for epitope recognition, contributing specific contacts at the center of the paratope in several anti-PAS complexes. Apart from these insights into how antibodies can recognize feature-less peptides without secondary structure, the MAbs characterized in this study offer valuable reagents for the preclinical and clinical development of PASylated biologics.
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http://dx.doi.org/10.1016/j.jmb.2021.167113DOI Listing
September 2021

First In-Human Medical Imaging with a PASylated Zr-Labeled Anti-HER2 Fab-Fragment in a Patient with Metastatic Breast Cancer.

Nucl Med Mol Imaging 2020 Apr 20;54(2):114-119. Epub 2020 Apr 20.

5Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.

Purpose: PASylation® offers the ability to systematically tune and optimize the pharmacokinetics of protein tracers for molecular imaging. Here we report the first clinical translation of a PASylated Fab fragment (Zr∙Df-HER2-Fab-PAS) for the molecular imaging of tumor-related HER2 expression.

Methods: A patient with HER2-positive metastatic breast cancer received 37 MBq of Zr∙Df-HER2-Fab-PAS at a total mass dose of 70 μg. PET/CT was carried out 6, 24, and 45 h after injection, followed by image analysis of biodistribution, normal organ uptake, and lesion targeting.

Results: Images show a biodistribution typical for protein tracers, characterized by a prominent blood pool 6 h p.i., which decreased over time. Lesions were detectable as early as 24 h p.i. Zr∙Df-HER2-Fab-PAS was tolerated well.

Conclusion: This study demonstrates that a PASylated Fab tracer shows appropriate blood clearance to allow sensitive visualization of small tumor lesions in a clinical setting.
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http://dx.doi.org/10.1007/s13139-020-00638-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198682PMC
April 2020

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

J Biol Chem 2020 01 9;295(3):868-882. Epub 2019 Dec 9.

Department of Medicine, University of Colorado, Aurora, Colorado 80045.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed -induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked potency and a significantly extended half-life compared with anakinra.
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http://dx.doi.org/10.1074/jbc.RA119.010340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970921PMC
January 2020

Regeneration After Radiation- and Immune-Mediated Tissue Injury Is Not Enhanced by Type III Interferon Signaling.

Int J Radiat Oncol Biol Phys 2019 03 29;103(4):970-976. Epub 2018 Nov 29.

Klinik und Poliklinik für Innere Medizin 3, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

Purpose: Type I interferon (IFN-I) and interleukin (IL)-22 modulate regeneration of the thymus and intestinal epithelial cells (IECs) after cytotoxic stress such as irradiation. Radiation-induced damage to thymic tissues and IECs is a crucial aspect during the pathogenesis of inadequate immune reconstitution and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with myeloablative total body irradiation (TBI), respectively. IL-22 and IFN-I reduce the severity of acute GVHD after allo-HSCT with myeloablative TBI. However, the role of biologically related type III interferon (IFN-III), also known as interferon lambda (IFN-λ) or IL-28, in this context is unclear. We therefore studied the role of the IFN-III pathway in thymic regeneration and GVHD after TBI and allo-HSCT.

Methods And Materials: Cohoused wild-type (WT) and IFN-III receptor-deficient (IL-28 receptor alpha subunit-deficient/IL-28Ra) mice were analyzed in models of TBI-induced thymus damage and a model of GVHD after allo-HSCT with myeloablative TBI. PASylated IFN-III (PASylated IL-28A, XL-protein GmbH) was generated to prolong the plasma half-life of IFN-III. Pharmacologic activity and the effects of PASylated IL-28A on radiation-induced thymus damage and the course of GVHD after allo-HSCT with myeloablative TBI were tested.

Results: The course and severity of GVHD after myeloablative TBI and allo-HSCT in IL-28Ra mice was comparable to those in WT mice. Activation of the IFN-III pathway by PASylated IL-28A did not significantly modulate GVHD after allo-HSCT with TBI. Furthermore, IL28Ra mice and WT mice showed similar thymus regeneration after radiation, which could also not be significantly modulated by IFN-III receptor engagement using PASylated IL-28A.

Conclusions: We analyzed the role of IFN-III signaling during radiation-mediated acute tissue injury. Despite molecular and biologic homologies with IFN-I and IL-22, IFN-III signaling did not improve thymus regeneration after radiation or the course of GVHD after myeloablative TBI and allo-HSCT.
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http://dx.doi.org/10.1016/j.ijrobp.2018.11.038DOI Listing
March 2019

PASylated interferon α efficiently suppresses hepatitis B virus and induces anti-HBs seroconversion in HBV-transgenic mice.

Antiviral Res 2019 01 12;161:134-143. Epub 2018 Nov 12.

Institute of Virology, Technische Universität München/Helmholtz Zentrum München, 81675, Munich, Germany; German Center for Infection Research (DZIF), Munich Partner Site, 81675, Munich, Germany. Electronic address:

Interferon α (IFNα) so far is the only therapeutic option for chronic hepatitis B virus (HBV) infection that can lead to virus clearance. Unfortunately, its application is limited by side effects and response rates are low. The aim of this study was to generate a novel long-acting IFNα with the help of PASylation technology that adds a polypeptide comprising Proline, Alanine and Serine (PAS) to increase plasma half-life. Following evaluation of four selected recombinant murine IFNα (mIFNα) subtypes in cell culture, the most active subtype, mIFNα11, was fused with a 600 amino acid PAS chain. The activity of PAS-mIFNα was assessed by interferon bioassay and further evaluated for induction of interferon-stimulated genes (ISG) and antiviral efficacy in cell culture as well as in HBV-transgenic mice. PAS-mIFNα induced expression of ISG comparable to unmodified mIFNα and, likewise, evoked dose-dependent reduction of HBV replication in vitro. In vivo, PAS-mIFNα led to pronounced suppression of HBV replication without detectable liver damage whereas conventional mIFNα treatment only had a modest antiviral effect. Importantly, all PAS-mIFNα treated mice showed an anti-HBs antibody response, lost HBsAg and achieved seroconversion after three weeks. PASylated IFNα showed a profoundly increased antiviral effect in vivo compared to the non-modified version without toxicity, providing proof-of-concept that an improved IFNα can achieve higher rates of HBV antiviral and immune control.
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http://dx.doi.org/10.1016/j.antiviral.2018.11.003DOI Listing
January 2019

Type I IFN signaling blockade by a PASylated antagonist during chronic SIV infection suppresses specific inflammatory pathways but does not alter T cell activation or virus replication.

PLoS Pathog 2018 08 24;14(8):e1007246. Epub 2018 Aug 24.

Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America.

Chronic activation of the immune system in HIV infection is one of the strongest predictors of morbidity and mortality. As such, approaches that reduce immune activation have received considerable interest. Previously, we demonstrated that administration of a type I interferon receptor antagonist (IFN-1ant) during acute SIV infection of rhesus macaques results in increased virus replication and accelerated disease progression. Here, we administered a long half-life PASylated IFN-1ant to ART-treated and ART-naïve macaques during chronic SIV infection and measured expression of interferon stimulated genes (ISG) by RNA sequencing, plasma viremia, plasma cytokines, T cell activation and exhaustion as well as cell-associated virus in CD4 T cell subsets sorted from peripheral blood and lymph nodes. Our study shows that IFN-1ant administration in both ART-suppressed and ART-untreated chronically SIV-infected animals successfully results in reduction of IFN-I-mediated inflammation as defined by reduced expression of ISGs but had no effect on plasma levels of IL-1β, IL-1ra, IL-6 and IL-8. Unlike in acute SIV infection, we observed no significant increase in plasma viremia up to 25 weeks after IFN-1ant administration or up to 15 weeks after ART interruption. Likewise, cell-associated virus measured by SIV gag DNA copies was similar between IFN-1ant and placebo groups. In addition, evaluation of T cell activation and exhaustion by surface expression of CD38, HLA-DR, Ki67, LAG-3, PD-1 and TIGIT, as well as transcriptome analysis showed no effect of IFN-I blockade. Thus, our data show that blocking IFN-I signaling during chronic SIV infection suppresses IFN-I-related inflammatory pathways without increasing virus replication, and thus may constitute a safe therapeutic intervention in chronic HIV infection.
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http://dx.doi.org/10.1371/journal.ppat.1007246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126880PMC
August 2018

PASylated Coversin, a C5-Specific Complement Inhibitor with Extended Pharmacokinetics, Shows Enhanced Anti-Hemolytic Activity in Vitro.

Bioconjug Chem 2016 10 26;27(10):2359-2371. Epub 2016 Sep 26.

Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München , Emil-Erlenmeyer-Forum 5, 85354 Freising (Weihenstephan), Germany.

The Ornithodoros moubata Complement Inhibitor (OmCI) binds complement component 5 (C5) with high affinity and, thus, selectively prevents proteolytic activation of the terminal lytic complement pathway. A recombinant version of OmCI (also known as Coversin and rEV576) has proven efficacious in several animal models of complement-mediated diseases and successfully completed a phase Ia clinical trial. Coversin is a small 17 kDa lipocalin protein which has a very short plasma half-life if not bound to C5; therefore, the drug requires frequent dosing. We have improved the pharmacokinetics of Coversin by N-terminal translational conjugation with a 600 residue polypeptide composed of Pro, Ala, and Ser (PAS) residues. To this end, PAS-Coversin as well as the unmodified Coversin were functionally expressed in the cytoplasm of E. coli and purified to homogeneity. Both versions showed identical affinity to human C5, as determined by surface plasmon resonance measurements, and revealed similar complement inhibitory activity, as measured in ELISAs with human serum. In line with the PEG-like biophysical properties, PASylation dramatically prolonged the plasma half-life of uncomplexed Coversin by a factor ≥50 in mice. In a clinically relevant in vitro model of the complement-mediated disease paroxysmal nocturnal hemoglobinuria (PNH) both versions of Coversin effectively reduced erythrocyte lysis. Unexpectedly, while the IC values were comparable, PAS-Coversin reached a substantially lower plateau of residual lysis at saturating inhibitor concentrations. Taken together, our data demonstrate two clinically relevant improvements of PASylated Coversin: markedly increased plasma half-life and considerably reduced background hemolysis of erythrocytes with PNH-induced phenotype.
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http://dx.doi.org/10.1021/acs.bioconjchem.6b00369DOI Listing
October 2016

Reprogramming the body weight set point by a reciprocal interaction of hypothalamic leptin sensitivity and Pomc gene expression reverts extreme obesity.

Mol Metab 2016 Oct 5;5(10):869-881. Epub 2016 Aug 5.

Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan Medical School, Ann Arbor, MI, USA. Electronic address:

Objective: A major challenge for obesity treatment is the maintenance of reduced body weight. Diet-induced obese mice are resistant to achieving normoweight once the obesogenic conditions are reversed, in part because lowered circulating leptin leads to a reduction in metabolic rate and a rebound of hyperphagia that defend the previously elevated body weight set point. Because hypothalamic POMC is a central leptin target, we investigated whether changes in circulating leptin modify Pomc expression to maintain normal energy balance in genetically predisposed obese mice.

Methods: Mice with reversible Pomc silencing in the arcuate nucleus (ArcPomc (-/-)) become morbidly obese eating low-fat chow. We measured body composition, food intake, plasma leptin, and leptin sensitivity in ArcPomc (-/-) mice weight-matched to littermate controls by calorie restriction, either from weaning or after developing obesity. Pomc was reactivated by tamoxifen-dependent Cre recombinase transgenes. Long acting PASylated leptin was administered to weight-reduced ArcPomc (-/-) mice to mimic the super-elevated leptin levels of obese mice.

Results: ArcPomc (-/-) mice had increased adiposity and leptin levels shortly after weaning. Despite chronic calorie restriction to achieve normoweight, ArcPomc (-/-) mice remained moderately hyperleptinemic and resistant to exogenous leptin's effects to reduce weight and food intake. However, subsequent Pomc reactivation in weight-matched ArcPomc (-/-) mice normalized plasma leptin, leptin sensitivity, adiposity, and food intake. In contrast, extreme hyperleptinemia induced by PASylated leptin blocked the full restoration of hypothalamic Pomc expression in calorie restricted ArcPomc (-/-) mice, which consequently regained 30% of their lost body weight and attained a metabolic steady state similar to that of tamoxifen treated obese ArcPomc (-/-) mice.

Conclusions: Pomc reactivation in previously obese, calorie-restricted ArcPomc (-/-) mice normalized energy homeostasis, suggesting that their body weight set point was restored to control levels. In contrast, massively obese and hyperleptinemic ArcPomc (-/-) mice or those weight-matched and treated with PASylated leptin to maintain extreme hyperleptinemia prior to Pomc reactivation converged to an intermediate set point relative to lean control and obese ArcPomc (-/-) mice. We conclude that restoration of hypothalamic leptin sensitivity and Pomc expression is necessary for obese ArcPomc (-/-) mice to achieve and sustain normal metabolic homeostasis; whereas deficits in either parameter set a maladaptive allostatic balance that defends increased adiposity and body weight.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034612PMC
http://dx.doi.org/10.1016/j.molmet.2016.07.012DOI Listing
October 2016

Treatment of diet-induced lipodystrophic C57BL/6J mice with long-acting PASylated leptin normalises insulin sensitivity and hepatic steatosis by promoting lipid utilisation.

Diabetologia 2016 09 7;59(9):2005-12. Epub 2016 Jun 7.

Lehrstuhl für Molekulare Ernährungsmedizin and Else Kröner-Fresenius Center, Technische Universität München, Gregor-Mendel-Str. 2, 85354, Freising (Weihenstephan), Germany.

Aims/hypothesis: Recombinant leptin offers a viable treatment for lipodystrophy (LD) syndromes. However, due to its short plasma half-life, leptin replacement therapy requires at least daily subcutaneous (s.c.) injections. Here, we optimised this treatment strategy in LD mice by using a novel leptin version with extended plasma half-life using PASylation technology.

Methods: A long-acting leptin version was prepared by genetic fusion with a 600 residue polypeptide made of Pro, Ala and Ser (PASylation), which enlarges the hydrodynamic volume and, thus, retards renal filtration, allowing less frequent injection. LD was induced in C57BL/6J mice by feeding a diet supplemented with conjugated linoleic acid (CLA). Chronic and acute effects of leptin treatment were assessed by evaluating plasma insulin levels, insulin tolerance, histological liver sections, energy expenditure, energy intake and body composition.

Results: In a cohort of female mice, 4 nmol PAS-leptin (applied via four s.c. injections every 3 days) successfully alleviated the CLA-induced LD phenotype, which was characterised by hyperinsulinaemia, insulin intolerance and hepatosteatosis. The same injection regimen had no measurable effect when unmodified recombinant leptin was administered at an equivalent dose. In a cohort of LD males, a single s.c. injection of PAS-leptin did not affect energy expenditure but inhibited food intake and promoted a shift in fuel selection towards preferential fat oxidation, which mechanistically substantiates the metabolic improvements.

Conclusions/interpretation: The excellent pharmacological properties render PASylated leptin an agent of choice for refining both animal studies and therapeutic strategies in the context of LD syndromes and beyond.
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http://dx.doi.org/10.1007/s00125-016-4004-6DOI Listing
September 2016

Long-Acting PASylated Leptin Ameliorates Obesity by Promoting Satiety and Preventing Hypometabolism in Leptin-Deficient Lep(ob/ob) Mice.

Endocrinology 2016 Jan 22;157(1):233-44. Epub 2015 Oct 22.

Lehrstuhl für Molekulare Ernährungsmedizin (F.B., A.B., N.R., S.M., M.K.), Else Kröner-Fresenius Center and ZIEL-Research Center for Nutrition and Food Science, and Munich Center for Integrated Protein Science and Lehrstuhl für Biologische Chemie (V.M., M.S., A.S.), Technische Universität München, 85350 Freising-Weihenstephan, Germany; and XL-protein GmbH (A.S.), 85354 Freising, Germany.

Body weight loss of Lep(ob/ob) mice in response to leptin is larger than expected from the reduction in energy intake alone, suggesting a thermogenic action of unknown magnitude. We exploited the superior pharmacological properties of a novel long-acting leptin prepared via PASylation to study the contribution of its anorexigenic and thermogenic effects. PASylation, the genetic fusion of leptin with a conformationally disordered polypeptide comprising 600 Pro/Ala/Ser (PAS) residues, provides a superior way to increase the hydrodynamic volume of the fusion protein, thus retarding kidney filtration and extending plasma half-life. Here a single PAS(600)-leptin injection (300 pmol/g) resulted in a maximal weight reduction of 21% 6 days after application. The negative energy balance of 300 kJ/(4 d) was driven by a decrease in energy intake, whereas energy expenditure remained stable. Mice that were food restricted to the same extent showed an energy deficit of only 220 kJ/(4 d) owing to recurring torpor bouts. Therefore, the anorexigenic effect of PAS(600)-leptin contributes 75% to weight loss, whereas the thermogenic action accounts for 25% by preventing hypometabolism. In a second experiment, just four injections of PAS(600)-leptin (100 pmol/g) administered in 5- to 6-day intervals rectified the Lep(ob/ob) phenotype. In total, 16 nmol of PAS(600)-leptin per mouse triggered a weight loss of 43% within 20 days and normalized hypothermia and glucose homeostasis as well as hepatic steatosis. The beneficial properties of PAS(600)-leptin are substantiated by a comparison with previous studies in which approximately 400 nmol (∼25-fold) unmodified leptin was mandatory to achieve similar improvements.
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http://dx.doi.org/10.1210/en.2015-1519DOI Listing
January 2016

PASylation of Murine Leptin Leads to Extended Plasma Half-Life and Enhanced in Vivo Efficacy.

Mol Pharm 2015 May 10;12(5):1431-42. Epub 2015 Apr 10.

∥XL-protein GmbH, Lise-Meitner-Strasse 30, 85354 Freising, Germany.

Leptin plays a central role in the control of energy homeostasis and appetite and, thus, has attracted attention for therapeutic approaches in spite of its limited pharmacological activity owing to the very short circulation in the body. To improve drug delivery and prolong plasma half-life, we have fused murine leptin with Pro/Ala/Ser (PAS) polypeptides of up to 600 residues, which adopt random coil conformation with expanded hydrodynamic volume in solution and, consequently, retard kidney filtration in a similar manner as polyethylene glycol (PEG). Relative to unmodified leptin, size exclusion chromatography and dynamic light scattering revealed an approximately 21-fold increase in apparent size and a much larger molecular diameter of around 18 nm for PAS(600)-leptin. High receptor-binding activity for all PASylated leptin versions was confirmed in BIAcore measurements and cell-based dual-luciferase assays. Pharmacokinetic studies in mice revealed a much extended plasma half-life after ip injection, from 26 min for the unmodified leptin to 19.6 h for the PAS(600) fusion. In vivo activity was investigated after single ip injection of equimolar doses of each leptin version. Strongly increased and prolonged hypothalamic STAT3 phosphorylation was detected for PAS(600)-leptin. Also, a reduction in daily food intake by up to 60% as well as loss in body weight of >10% lasting for >5 days was observed, whereas unmodified leptin was merely effective for 1 day. Notably, application of a PASylated superactive mouse leptin antagonist (SMLA) led to the opposite effects. Thus, PASylated leptin not only provides a promising reagent to study its physiological role in vivo but also may offer a superior drug candidate for clinical therapy.
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http://dx.doi.org/10.1021/mp5007147DOI Listing
May 2015

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

MAbs 2015 ;7(1):96-109

a Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie ; Technische Universität München ; Freising-Weihenstephan , Germany.

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.
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http://dx.doi.org/10.4161/19420862.2014.985522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622060PMC
September 2015

Enhanced in vivo efficacy of a type I interferon superagonist with extended plasma half-life in a mouse model of multiple sclerosis.

J Biol Chem 2014 Oct 5;289(42):29014-29. Epub 2014 Sep 5.

From the Departments of Biological Chemistry,

IFNβ is a common therapeutic option to treat multiple sclerosis. It is unique among the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNSα8) that is built on the backbone of a low affinity IFNα but modified to exhibit higher receptor affinity than even for IFNβ. Here, YNSα8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain comprising proline, alanine, and serine (PAS) to prolong its plasma half-life via "PASylation." PAS-YNSα8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors, notably without any detectable loss in biological potency or bioavailability. This long-lived superagonist conferred significantly improved protection from MOG35-55-induced experimental autoimmune encephalomyelitis compared with IFNβ, despite being injected with a 4-fold less frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS measurements showing a decrease of CD11b(+)/CD45(hi) myeloid lineage cells detectable in the CNS, as well as a decrease in IBA(+) cells in spinal cord sections determined by immunohistochemistry for PAS-YNSα8-treated animals. Importantly, PAS-YNSα8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. A striking correlation between increased levels of CD274 (PD-L1) transcripts from spleen-derived CD4(+) cells and improved clinical response to autoimmune encephalomyelitis was observed, indicating that, at least in this mouse model of multiple sclerosis, CD274 may serve as a biomarker to predict the effectiveness of IFN therapy to treat this complex disease.
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http://dx.doi.org/10.1074/jbc.M114.602474DOI Listing
October 2014

PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins.

Protein Eng Des Sel 2013 Aug 10;26(8):489-501. Epub 2013 Jun 10.

Munich Center for Integrated Protein Science CIPS-M & Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85350 Freising-Weihenstephan, Germany.

A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.
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http://dx.doi.org/10.1093/protein/gzt023DOI Listing
August 2013

Intravenous application of an anticalin dramatically lowers plasma digoxin levels and reduces its toxic effects in rats.

Toxicol Appl Pharmacol 2012 Sep 20;263(3):352-9. Epub 2012 Jul 20.

Department of Toxicology, Klinikum rechts der Isar, Munich, Germany.

Lipocalins tailored with high affinity for prescribed ligands, so-called anticalins, constitute promising candidates as antidotes. Here, we present an animal study to investigate both pharmacokinetic and clinical effects of an anticalin specific for the digitalis compound digoxin. Intravenous digoxin (2.5-50 μg/kg/min) was administered to rats until first changes in the ECG occurred (dose finding study) or a priori for 30 min (kinetic study). The anticalin DigA16(H86N), dubbed DigiCal, was administered intravenously at absolute doses of 1, 5, 10 and 20 mg, while the control group received isotonic saline. Hemodynamic changes, several ECG parameters and digoxin concentration in plasma were monitored at given time intervals. After DigiCal administration free digoxin concentration in plasma ultrafiltrate declined dramatically within 1 min to the presumably non-toxic range. There was also a significant and DigiCal dose-dependent effect on longer survival, less ECG alterations, arrhythmia, and improved hemodynamics. Infusion of a lower digoxin dose (2.5 μg/kg/min) resulted in a more sustained reduction of free digoxin in plasma after DigiCal administration compared to a higher digoxin dose (25 μg/kg/min), whereas ECG and hemodynamic parameters did not markedly differ, reflecting the known relative insensitivity of rats towards digoxin toxicity. Notably, we observed a re-increase of free digoxin in plasma some time after bolus administration of DigiCal, which was presumably due to toxin redistribution from tissue in combination with the relatively fast renal clearance of the rather small protein antidote. We conclude that anticalins with appropriately engineered drug-binding activities and, possibly, prolonged plasma half-life offer prospects for next-generation antidotal therapy.
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http://dx.doi.org/10.1016/j.taap.2012.07.009DOI Listing
September 2012

Novel serial positive enrichment technology enables clinical multiparameter cell sorting.

PLoS One 2012 24;7(4):e35798. Epub 2012 Apr 24.

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high)/CD25(high)/CD45RA(high) 'regulatory T cells' and CD8(high)/CD62L(high)/CD45RA(neg) 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035798PLOS
August 2012

High molecular mass radioimmunoconjugates are promising for intraperitoneal α-emitter immunotherapy due to prolonged retention in the peritoneum.

Nucl Med Biol 2012 Jul 3;39(5):617-27. Epub 2012 Mar 3.

Department of Nuclear Medicine, Technische Universität München, 81675 Munich, Germany.

Introduction: Therapeutic efficacy of intraperitoneal radioimmunotherapy is dependent on the time of retention of the radioimmunoconjugates within the peritoneal cavity. Therefore, the aim of this study was to investigate intraperitoneal retention of Fab, IgG and IgM radioimmunoconjugates.

Methods: Female Balb/c mice were injected with 213Bi- or 111In-labeled IgM, IgG and recombinant Fab conjugates intraperitoneally or intravenously. At different time points after injection, whole body distribution of radionuclides was imaged using a gamma camera. Distribution of radionuclides in selected organs was determined via γ-counting after sacrifice. Biological half-lives of the conjugates were calculated from whole body activities.

Results: After i.p. injection 213Bi-Fab rapidly accumulated in the kidneys indicative of glomerular filtration and reabsorption. Accumulation of 213Bi-IgG in the kidneys was significantly lower. 213Bi-IgM showed a striking accumulation in the liver 180 min after i.p. injection. 111In-IgG persisted in the circulation up to 72 h both after i.p. and i.v. injection. 111In-IgM showed a continuous accumulation in the liver. Moreover, 111In-IgM was significantly higher 24 h after i.v. injection than i.p. injection both in liver and spleen. These differences could be confirmed via scintigraphy. After injection of 111In-IgG differences in scintigraphic images between i.v. and i.p. were clearly visible only at 3 h. Biological half lives were 24 h, 45 h and 165 h for 111In-IgM, 111In-Fab and 111In-IgG, respectively.

Conclusions: Retention of radioimmunoconjugates in the peritoneal cavity positively correlates with the molecular mass of the antibody. Therefore, IgM radioimmunoconjugates should be preferably used in radioimmunotherapy of free floating tumor cells and small tumor cell clusters in the ascites of the peritoneal cavity.
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http://dx.doi.org/10.1016/j.nucmedbio.2011.12.005DOI Listing
July 2012

Periplasmic chaperones used to enhance functional secretion of proteins in E. coli.

Methods Mol Biol 2011 ;705:211-24

Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany.

While Escherichia coli is in wide use as a host organism for preparative protein production, problems with the folding of the recombinant gene product as well as protein aggregation, i.e., formation of inclusion bodies, are frequently encountered. This is particularly true for proteins that carry structural disulfide bonds, including antibody fragments, cytokines, growth factors, and extracellular fragments of eukaryotic cell surface receptors. In these cases, secretion into the oxidizing milieu of the bacterial periplasm in principle enables disulfide bond formation, resulting in a correctly folded and soluble protein. However, this process often occurs at low efficiency, depending on the nature of the recombinant gene product. Therefore, we have developed the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and/or folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC, which catalyze the formation and isomerization of disulfide bridges, and two peptidyl-prolyl cis/trans isomerases with chaperone activity, FkpA and SurA. Here, we present a detailed protocol how to use this system for the bacterial secretion of recombinant proteins, including human EGF as a new example, and we give hints on optimization of the expression procedure.
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http://dx.doi.org/10.1007/978-1-61737-967-3_12DOI Listing
March 2011

Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains.

Protein Eng Des Sel 2009 Mar 19;22(3):175-88. Epub 2008 Nov 19.

Lehrstuhl für Biologische Chemie, Technische Universität München, Germany.

An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.
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http://dx.doi.org/10.1093/protein/gzn066DOI Listing
March 2009

Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life.

Protein Eng Des Sel 2007 Jun 26;20(6):273-84. Epub 2007 Jun 26.

Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.

Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly(4)Ser)(n) to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His(6) tag fused to the heavy chain and the Strep-tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.
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http://dx.doi.org/10.1093/protein/gzm020DOI Listing
June 2007

A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli.

Protein Eng Des Sel 2006 Aug 23;19(8):385-90. Epub 2006 May 23.

Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.

Although Escherichia coli is in wide use for preparative protein expression, problems with the folding of the recombinant gene product and protein aggregation are frequently encountered. Apart from cytoplasmic expression, this is also true for secretion into the bacterial periplasm, the method of choice for the production of proteins that carry structural disulfide bonds. Here we report the construction of the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC that catalyze the formation and isomerization of disulfide bridges and the peptidyl-prolyl cis/trans-isomerases with chaperone activity, FkpA and SurA. pTUM4 carries a p15a origin of replication and a chloramphenicol resistance gene and, thus, it is compatible with many conventional expression vectors that use the ColEI origin and an ampicillin resistance. Its positive effects on the yield of soluble recombinant protein and the homogeneity of disulfide pattern are illustrated here using the human plasma retinol-binding protein as well as the extracellular carbohydrate recognition domain of the dendritic cell membrane receptor DC-SIGN. Hence, pTUM4 represents a novel helper vector which complements existing cytosolic chaperone coexpression plasmids and should be useful for the functional secretion of various recombinant proteins with hampered folding efficiency.
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http://dx.doi.org/10.1093/protein/gzl018DOI Listing
August 2006

Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach.

Protein Eng Des Sel 2004 Dec 11;17(12):847-60. Epub 2005 Feb 11.

Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan and CR/MP Saarland University Medical School, 66421 Homburg/Saar, Germany.

CD30, the so-called Reed-Sternberg antigen, constitutes a promising cell-specific target for the treatment of Hodgkin's lymphoma. Starting from the previously characterized cognate HRS3 mouse monoclonal antibody, the bacterially produced functional Fab fragment was humanized by grafting the CDRs from the mouse antibody framework on to human immunoglobulin consensus sequences. This procedure led to a 10-fold decreased antigen affinity, which surprisingly was found to be mainly due to the VH domain. To improve the antigen-binding activity, an in vitro evolution strategy was employed, wherein random mutations were introduced into the humanized VH domain by means of error-prone PCR, followed by a filter sandwich Escherichia coli colony screening assay for functional Fab fragments using a recombinant extracellular domain of the CD30 antigen. After three cycles of in vitro affinity maturation, the optimized Fab fragment huHRS3-VH-EP3/1 was identified, which carried four exchanged residues within or close to the VH CDRs and had an affinity that was almost identical with that of the murine HRS3 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to CD30 binding both in ELISA with the recombinant antigen and in FACS experiments with CD30-positive L540CY cells. In the light of the previously successful clinical application of an alphaCD30 x alphaCD16 bispecific mouse quadroma antibody derived from HRS3, the humanized Fab fragment comprises an important step towards the construction of a fully recombinant therapeutic agent. The combination of random mutagenesis and colony filter screening assay that was successfully applied here should be generally useful as a method for the rapid functional optimization of humanized antibody fragments.
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http://dx.doi.org/10.1093/protein/gzh098DOI Listing
December 2004

The periplasmic E. coli chaperone Skp is a trimer in solution: biophysical and preliminary crystallographic characterization.

Biol Chem 2004 Feb;385(2):137-43

Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany.

The 'seventeen kilodalton protein' Skp confers transient solubility on outer membrane proteins during biogenesis in Gram-negative bacteria. Here we report a first biophysical characterization of this chaperone itself, which also possesses biotechnological potential in the production of recombinant proteins. Using cross-linking and gel filtration methods, we found that Skp forms a stable homo-trimer in solution. Following thermal denaturation, monitored by CD spectroscopy, this chaperone refolds with high efficiency but exhibits a pronounced hysteresis between the un- and refolding transitions. Using the recombinant protein equipped with the Strep-tag II at its N-terminus, suitable crystallization conditions for Skp were found. A first data set was collected to 2.60 A resolution.
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http://dx.doi.org/10.1515/BC.2004.032DOI Listing
February 2004
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