Production of biologically active recombinant human factor H in Physcomitrella

Abstract

SummaryThe human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry.Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma.

An Engineered Construct Combining Complement Regulatory and Surface-Recognition Domains Represents a Minimal-Size Functional Factor H

“…FH is a large molecule with multiple cysteine bridges; therefore, its recombinant production is difficult and expensive, although production of recombinant full-length FH was recently reported (49,50). In this study, we describe mini-FH, a compact form of FH limited to the two main functional regions, as a potent complement inhibitor.…”

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens

“…The knock-in construct for the generation of Cerulean:sHSP was transfected into the background of a stable ATE:Citrine knock-in line (#49), as well as into the background of a stable ATE:GUS knock-in line (#9) (10), using a co-transfected hygromycin b phosphotransferase (hpt) cassette as transient selection marker (52,53) (hpt cassette in pJET1.2, Thermo Scientific), selection was performed on Knop-ME medium with 12.5 mg/l hygromycin). Integration of the knock-in construct at the target locus was validated by PCR on genomic DNA of the transgenic lines using primers that annealed to the genomic sequence upstream and downstream of the homologous regions of the knock-in construct, respectively (see supplemental Coimmunoprecipitation (CoIP) of Interaction Partners of ATE-Six grams fresh weight of gametophores from ATE:GUS and wild type were harvested from hydroponic ring cultures after 7 d cultivation in darkness and used for CoIP.…”

Clinical Evaluation of 3 Families With Basal Laminar Drusen Caused by Novel Mutations in the Complement Factor H Gene

“…This manifests as a loss of function of CFH, resulting in increased activity of the complement system's alternative pathway. [47][48][49] Humanized monoclonal antibodies can inhibit the overactivated complement system. For this reason, humanized monoclonal antibody seems to be a rational candidate treatment for patients with BLD caused by mutations in the CFH gene.…”