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Expression, purification and characterization of recombinant caprine N-acetylglucosamine-6-sulphatase.

ABSTRACT: Mucopolysaccharidosis type IIID or Sanfilippo D syndrome is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulphatase (Glc6S). In addition to human patients, a Nubian goat with this disorder has been described and the caprine Glc6S (cGlc6S) cDNA cloned. In this study, the full-length cGlc6S cDNA was inserted into the expression vector, pEFNeo, which placed the cGlc6S cDNA under the transcriptional control of the human polypeptide chain elongation factor promoter. The pEFNeo expression vector also contains the human growth hormone polyadenylation signal and the genes encoding resistance to ampicillin and G418. The cGlc6S expression construct was electroporated into Chinese hamster ovary (CHO-K1) cells, and stably transfected clones were isolated. One clone, CHOrcGlc6S.17, which secreted the highest Glc6S activity into the culture medium, was selected and cultured in cell factories. The secreted recombinant cGlc6S (rcGlc6S) precursor was purified to homogeneity from conditioned medium by a two-column procedure which consisted of a Cu2+-chelating Sepharose column followed by TSK G3000SW gel filtration. The native molecular mass of rcFlc6S was estimated to be 102 kDa and the subunit size was 94 kDa. The kinetic properties of cGlc6S were similar to those of human Glc6S isolated from liver. rcGlc6S was endocytosed by fibroblasts from patients with mucopolysaccharidosis type IIID via the mannose 6-phosphate receptor-mediated pathway resulting in correction of the storage phenotype of these cells.

SUBMITTER: Litjens T

PROVIDER: S-EPMC1218767 | biostudies-other | 1997 Oct

REPOSITORIES: biostudies-other

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Project description:The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2?26. eGFP-Kir6.2?26 was expressed in HEK293?cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2?26 retains native single channel conductance (64?±?3.3?pS), mean open times (?1?=?0.72?ms, ?2?=?15.3?ms) and ATP affinity (IC50?=?115?±?25??M) when expressed in HEK293?cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co2+-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2?26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), l-?-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5?mol ratio. Reconstituted eGFP-Kir6.2?26 displayed similar single channel conductance (61.8?±?0.54?pS) compared to eGFP-Kir6.2?26 expressed in HEK293 membranes; however, channel mean open times increased (?1?=?7.9?ms, ?2?=?61.9?ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2?26 reconstituted into BLMs (IC50?=?3.14?±?0.4?mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.

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Expression, purification and characterization of recombinant caprine N-acetylglucosamine-6-sulphatase.

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