Enzyme replacement therapy for mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo B

Enzyme replacement therapy for mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo B syndrome) has been hindered by inadequate mannose 6-phosphorylation and cellular uptake of recombinantly produced human α-and pH optimum of the fusion enzyme was much like those reported for rhNAGLU. hampering their ability to cross-correct tissues away from the injection and implantation sites. The M6P receptor also binds insulin-like growth factor-II and is henceforth referred to as the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR) [23]. Here we have employed a fusion protein strategy to enable recombinantly produced NAGLU to gain access to cells through M6P/IGF-IIR which recognizes multiple ligands on its extracellular domain name including M6P-containing lysosomal enzymes retinoic acid and IGF-II via unique binding sites around the receptor surface [24 25 Previous work by others showed that fusion proteins of lysosomal enzymes and IGF-II joined cells and lysosomes via the M6P/IGF-IIR around the cell membrane [26 27 We generated an expression construct of and the receptor binding domain name of around the C-terminus to produce recombinant human NAGLU-IGF-II (rhNAGLUIGF-II). In this study rhNAGLU-IGF-II was expressed and purified from Chinese hamster ovary (CHO) cells for biochemical characterization and further tested for Luseogliflozin functional delivery to MPS IIIB Luseogliflozin cells and brain tumor-derived cell lines and correction of GAG storage (“type”:”entrez-nucleotide” attrs :”text”:”NM_000263.3″ term_id :”66346697″ term_text :”NM_000263.3″NM_000263.3) a short unstructured linker and the c-myc epitope (EQKLISEED) followed by a portion of the cDNA (“type”:”entrez-nucleotide” attrs :”text”:”NM_001007139.4″ term_id :”183603938″ term_text :”NM_001007139.4″NM_001007139.4) encoding amino acids 32-91 was synthesized using codon optimization for expression in CHO cells by Genscript USA Inc (Piscataway NJ) and provided in the cloning vector pUC57. The DNA fragment was subcloned into pCI-neo (Promega Rabbit polyclonal to ZNF483. Corporation Madison WI) at the cDNA was provided in pCMV-NAGLU by Dr. E. F. Neufeld (University or college of California Los Angeles CA) and was subcloned into pCI-neo at the cells were cultured in Ham’s F12/DME (Irvine Scientific Irvine CA) supplemented with 10% fetal bovine serum (SAFC Biosciences Lenexa KS) 1 mM non-essential amino acids 1 mM sodium pyruvate 2 mM L-glutamine and antibiotics (100 models/ml penicillin G 100 μg/ml streptomycin sulfate Irvine Scientific Irvine CA) and 50 μg/ml gentamicin sulfate (EMD Chemicals Inc. Gibbstown NJ) at 37°C in a 5% CO2 air flow atmosphere. Luseogliflozin pCI-NagGScIGF (or pCINAGLU) was linearized with I (New England BioLabs Ipswich MA) and transfected into CHO cells using PolyFect Transfection Reagent (Qiagen Inc. Valencia CA). The stable lines were selected by their resistance to 700 μg/ml G-418 (EMD Chemicals Inc. Gibbstown NJ) and colonies were created after 7-14 days. Individual colonies were isolated and the highestyielding expressors of secreted rhNAGLU-IGF-II (5H10) or rhNAGLU (117-1.511) were identified by NAGLU activity assay. Stable CHO cell clones were managed in Ham’s F12/DME with supplements made up of 250 μg/ml G-418. For protein production rhNAGLU-IGF-II clone 5H10 or rhNAGLU clone 117-1.511 were seeded into roller bottles and grown to confluence at which time the medium was replaced with EX-CELL PF CHO serum-free medium supplemented with 4 mM L-glutamine nucleosides (10 mg/l each of guanosine adenosine uridine cytosine hypoxanthine and thymidine) 50 μg/ml gentamicin sulfate and 250 μg/ml G-418. Secreted NAGLU activity Luseogliflozin was monitored daily for 7-14 days until NAGLU expression reached a plateau before the conditioned medium was harvested for enzyme purification. Purification of altered recombinant NAGLU enzymes rhNAGLU-IGF-II and rhNAGLU enzymes were purified from culture medium. Conditioned medium was filtered (0.2 μm) supplemented with methyl-α-d-glucopyranoside (10 mM) and stored at 4°C prior to purification (below). Medium containing rhNAGLU-IGF-II was initially concentrated using an Amicon ultrafiltration concentrator with a YM30 membrane Luseogliflozin (EMD Millipore Corp. Billerica MA) to its 20% volume dialyzed against PBS and loaded onto an 80 ml Concavalin A (Con A) Sepharose column (GE Healthcare Bio-Sciences Corp Piscataway NJ) pre-equilibrated with binding buffer (20 mM sodium phosphate pH 6.8; 300 Luseogliflozin mM NaCl; 10 mM methyl-α-d-glucopyranoside; 1 mM β- mercaptoethanol). The column was washed with one.