The 1.5-kb NdeI-BamHI fragment carrying desV and the 1.9-kb NdeI-XhoI fragment carrying ligV from pT21-2832 and pLVH, respectively, were ligated into the corresponding sites of pET-16b. The resultant plasmids, pT16-desV and pT16-ligV, were independently introduced into E. coli BL21(DE3), and then the His tag-fused desV and ligV were expressed. For purification, cell extracts were applied to His GraviTrap TALON columns (GE Healthcare). Purified fractions were subjected to desalting and centrifugal filtration with Amicon Ultra 30k (Millipore), and stored at −30 °C until use. The purity of the preparations was examined by SDS–12% PAGE.
His gravitrap talon columns
Manufactured by GE Healthcare
The GraviTrap TALON columns are a type of affinity chromatography resin designed for the purification of His-tagged proteins. They utilize a cobalt-based metal chelate to capture and separate the target proteins from complex mixtures. The columns provide a simple and efficient method for protein purification.
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Lab products found in correlation
2 protocols using his gravitrap talon columns
1
Purification of DESVand LIGVProteins
2
Incorporating Non-Canonical Amino Acids
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The gene of monomeric neon-green Green fluorescent protein (nGFP) in a plasmid with CloDF13 origin of replication and amber stop codon interrupted at position 13 was used to test the incorporation of non-canonical amino acids. 'Wild type' (WT) neon-green monomeric GFP was expressed from a pET vector with pBR322 origin of replication. A derivative Methanococcus jannaschii Tyrosyl aminoacyl-tRNA synthetase (aaRS) and tRNAsup pair was used for incorporation of para-acetyl-phenylalanine(PaF). 27 This aaRS/tRNAsup pair is under a GlnS promoter in a plasmid with pUC origin of replication. The same construct with Methanosarcina mazei Pyrrolysl tRNA aaRS/tRNAsup was used to incorporate N ε -(tert-butoxycarbonyl)-L-lysine (boc-lysine) into both nGFP and PpiB, at positions A13TAG and A147TAG respectively.
For protein purification, 1 ml His GraviTrap TALON columns (GE Healthcare) were used for PPiB, which was expressed in an encapsulated pellet from 500ml of culture, 3mls of CFPS to induce expression and had approximately 0.5mg/ml of boc-lysine incorporated PPiB in 500μl. For photocaged cysteine incorporation, cells were cultured in 1L and then encapsulated. The volume of CFPS buffer was 30mL and the protein yield 0.65mg. Protein concentration was determined using the Thermo Scientific Nanodrop™ One/Onec Microvolume UV spectrophotometer.
For protein purification, 1 ml His GraviTrap TALON columns (GE Healthcare) were used for PPiB, which was expressed in an encapsulated pellet from 500ml of culture, 3mls of CFPS to induce expression and had approximately 0.5mg/ml of boc-lysine incorporated PPiB in 500μl. For photocaged cysteine incorporation, cells were cultured in 1L and then encapsulated. The volume of CFPS buffer was 30mL and the protein yield 0.65mg. Protein concentration was determined using the Thermo Scientific Nanodrop™ One/Onec Microvolume UV spectrophotometer.
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