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Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP
KTH, School of Biotechnology (BIO), Protein Technology. (HPR/M Uhlén)
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0001-8993-048X
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2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 11, e81350- p.Article in journal (Refereed) Published
Abstract [en]

Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.

Place, publisher, year, edition, pages
2013. Vol. 8, no 11, e81350- p.
Keyword [en]
I-Related Receptor, Ph-Dependent Binding, Immunoglobulin-G, Crystal-Structure, Functional Expression, Igg, Complex, Cells, Generation, Antibodies
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-136198DOI: 10.1371/journal.pone.0081350ISI: 000327308500205PubMedID: 24260574ScopusID: 2-s2.0-84894137752OAI: diva2:675566
Knut and Alice Wallenberg Foundation

QC 20140613

Available from: 2013-12-04 Created: 2013-12-04 Last updated: 2014-06-13Bibliographically approved
In thesis
1. Rational and Combinatorial Engineering of Affinity Proteins Towards Therapeutical Applications
Open this publication in new window or tab >>Rational and Combinatorial Engineering of Affinity Proteins Towards Therapeutical Applications
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein engineering has had an immense impact on the development of biological drugs, including replacement therapies with engineered versions of insulin or factor VIII to treat diabetes or bleeding disorders, and monoclonal antibodies to treat cancer and various other malignancies. Now, with the next generation of treatment modalities coming up, including monoclonal reagents based on alternative scaffolds, gene and cell therapies, the importance of protein engineering to tailor-make these treatments is likely to increase further.

The neonatal Fc receptor (FcRn) is widely expressed in the body. One of the receptor's interesting functions is to rescue immunoglobulin G (IgG) and serum albumin (SA) from degradation by cells in contact with blood. When serum proteins are endocytosed by a cell, they are transported via the endosome to the lysosome for degradation. However, IgG and SA associate with the FcRn already at the slightly acidic pH of the endosome followed by transport back to the cell surface. There the complex encounters the neutral pH of the blood, at which the binding affinity to FcRn is lost, and IgG and SA are released back into circulation. In the main part of this thesis, efforts are described aiming to take use of, or block, the FcRn recycling mechanism to control the serum circulation half-life of proteins.

In the first study (Study I), a robust expression strategy for human FcRn was designed and evaluated. The extracellular domain was produced in the human SKOV3 cell-line after facile lentiviral delivery of the expression cassettes. This lead to continuous expression of secreted protein that could be purified to homogeneity by a single affinity chromatographic step, using the intrinsic pH-dependent interaction between FcRn and IgG, where the latter was immobilized in a column. The amount of purified protein was 1.4 mg per liter medium. The protein was characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay. The results suggested a fully functional and stable protein of high purity. In addition, the gene encoding full-length FcRn as a fusion to eGFP was delivered to HeLa cells utilizing the same lentiviral system. Subsequent analysis by flow cytometry and confocal fluorescence microscopy indicated a wide distribution of eGFP/FcRn expression among the cells. Binding of IgG and HSA was found to correlate well with the amount of eGFP/FcRn expressed by the cells.

In a following study (Study II), the goal was to generate affinity proteins interacting with human FcRn in a pH-dependent manner similar to that of FcRn's natural ligands. The affinity proteins used are denoted affibody molecules, a class of small alternative scaffold proteins with a three-helical structure. Affibody molecules were selected from a combinatorial library displayed on phage where binding took place at pH 5.5 and elution was performed at pH 2.2 or 8.0. Selected variants were characterized by developed in vitro and cell based assays, and some were found to have the desired pH-dependent binding to FcRn. In vivo studies in mice showed that the serum half-life of a model protein, genetically fused to the FcRn binding affibody molecules, was extended up to almost three-fold compared to a control protein (from 33 to 91 hours).

In a subsequent study (Study III), the use of a FcRn binding affibody molecule to reduce, rather than prolong, the serum half-life of proteins was explored. Here, the rationale was to investigate if injection of a FcRn binding affibody could hinder endogenous IgG from being rescued by FcRn, which could lead to depletion of IgGs by lysosomal degradation. In autoimmune diseases, such depletion of IgG would include also pathogenic IgG and could thus mitigate the symptoms of the disease.  Using cell based assays, it was found that one affibody molecule, selected in Study II, could readily block IgG from binding both human and murine FcRn. A following in vivo study in mice showed that systemic injection of the molecule reduced the amount of endogenous IgG by 39%.

In a fourth study (Study IV), the goal was to use a different class of affinity proteins to regulate the level of an enzyme in the brain. More specifically, artificial zinc finger-based transcription factors regulating the level of GAD67, which is the rate-limiting enzyme in synthesis of gamma-aminobutyric acid (GABA), were designed. Imbalances in GABA-signaling is involved in different diseases, including Parkinson's disease and epilepsy, and regulation of GAD67 at particular sites in the brain might be a route to ameliorate symptoms associated with such diseases. ELISA-based investigation showed that one of the designed zinc fingers, denoted G3, bound selectively to its intended target DNA sequence. A construct encoding G3 fused to a general transcriptional activator (VP64) was delivered to PC12-cells, using a lentivirus-based gene delivery system, resulting in a significant up-regulation of endogenous GAD67 expression. The same construct was subsequently delivered to the striatum of rats, with an induced disease model of Parkinson's disease. Western blot of striatal samples showed a significant up-regulation of GAD67 expression in lesioned striatum compared to intact striatum, and a tendency towards up-regulation compared to lesioned striatum.

Taken together, the protein engineering efforts described in this thesis concerning affinity proteins binding other proteins or DNA, has the potential to find use in drug development and may benefit patients in the future.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. 113 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:5
neonatal Fc receptor, FcRn, affibody molecule, immunoglobulin G (IgG), serum albumin (SA), albumin binding domain (ABD), half-life extension, blocking, autoimmune disease, pH-dependence, GAD67, GAD-1, gene therapy, Parkinson’s disease, zinc finger based artificial transcription factor
National Category
Medical Biotechnology Biochemistry and Molecular Biology
Research subject
urn:nbn:se:kth:diva-144297 (URN)978-91-7595-066-2 (ISBN)
Public defence
2014-05-23, FR4, AlbaNova, Roslagstullsbacken 21, 114 21 Stockholm, 10:00 (English)
Knut and Alice Wallenberg Foundation

QC 20140417

Available from: 2014-04-17 Created: 2014-04-16 Last updated: 2014-07-15Bibliographically approved

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