Preparation Of GSH Affinity Chromatographic Adsorbents And Their Application On Purification Of GST-Tagged Protein

The GST tag is inserted into an expression vector and can express in E. coli expression system. Because of the specific binding of GST to GSH, the GST fused proteins can be purified effectively by GSH affinity chromatography. The Glutathione (GSH) affinity adsoebent coupled with GSH was prepared with GE Healthcare Sepharose 4 Fast Flow and 1,4-butanediol diglycidyl ether (BDGE). The activating and coupling conditions of agarose particles were optimized with the Gultathione S transferase (GST), the recombinant GST-BCl-2, GST-PTPN12 and GST-Ald proteins.0.42 g NaOH,15 mL DMSO and 15 mL BDGE were added into 10 mL Sepharose 4 Fast Flow and then incubated at 40 ℃ for 6 h. The density of epoxy group of agarose particles was about 60-65 μmol/mL. The loading capacity was optimized to be about 14.40±0.77 mg/mL with coupling reaction solution pH 6.5.Alanine dehydrogenase (Ald, EC 1.4.1.1) is an amino acid dehydrogenases that catalyzes the NADH-dependent reversible conversion of pyruvate to L-alanine. The crystal structure offer more favorable conditions for expounding the catalytic mechanism of the enzyme protein.The gene Ald encoding l-AlaDH protein (GenBank accession No.345442329) was amplified from Bacillus. megaterium WSH-002 genome DNA using . the primers containing restriction sites. The amplified PCR products were digested with corresponding restriction enzyme and subsequently inserted into pET-28a(+) and pGEX-6P-1 (Novagen, USA). Escherichia coli BL21(DE3) strain was transformed with the two vectors respectively. The soluble expression was obtained when induced by IPTG at 16 ℃. The supernatant was filtered and loaded onto a Ni2+-chelating affinity chromatography column (GE Healthcare, USA) or a GSH affinity chromatography column of a lab-made. The eluted Ald protein was concentrated and further purified by size-exclusion chromatography on a Superdex 75 10/300 GL column (GE Healthcare, USA). The purity of the protein was analysed by 12% SDS-PAGE. The recombinant plasmids pGEX-6P-1/Ald and pET-28a-Ald purified the recombinant protein for crystallization screening. For the protein purified from pGEX-6P-1/Ald, the optimum growing condition of the crystal consisted of 0.03 M citric acid pH 7.5,16%(w/v) polyethylene glycol 3350 at the protein purified from concentration of 12 mg/mL. The crystal grew in a solution consisting of 0.1 M HEPES pH 8.0,12%(w/v) polyethylene glycol 8000,8%(v/v) ethylene glycol at the concentration of 15 mg/mL with protein acquired from pET-28a-Ald. The crystals all grew with good reproducibility in this solution and X-ray diffraction data were collected to 2.22 A resolution (pGEX-6P-1/Ald) and 2.35 A resolution (pET-28a-Ald). The crystals belonged to trigonal space group R32, with only one protein molecule in an asymmetric unit. Sequence alignment with known structures in the PDB showed that Ald from B. megaterium shares 62% sequence similarity with that from Thermus thermophilus (PDB code 2eez). A search model for MR calculations was generated from 2eez in which non-identical amino-acid residues were replaced by alanine.