Development of a novel Gateway-based vector system for efficient, multiparallel protein expression in Escherichia coli

para-Aminosalicylic Acid Is a Prodrug Targeting Dihydrofolate Reductase in Mycobacterium tuberculosis

“…Protein Purification and Analysis-M. tuberculosis H37Rv dfrA, folP (DHPS), and ribD were expressed as His 6 -tagged proteins in Escherichia coli BL21 as described previously and purified by standard procedures (13). Proteins were analyzed with Western blot.…”

Preliminary crystallographic analysis of the kinase domain of SAD-1, a protein essential for presynaptic differentiation inCaenorhabditis elegans

“…After concentration using an Amicon Ultra filter (Millipore), the fusion protein was dissolved in low-salt buffer (20 mM Tris, 20 mM NaCl pH 7.5). The Trx-6ÂHis-ceSKD protein was then digested using PreScission protease (Freuler et al, 2008) at 277 K for 12 h and the cleaved protein, which had four additional residues (GPGS) at its N-terminus, was purified on a HiTrap SP HP column (GE Healthcare) using a linear NaCl gradient from 20 to 800 mM. The protein peak corresponding to ceSKD was pooled, concentrated, loaded onto a HiLoad 16/60 Superdex 200 pg column (GE Healthcare Life Sciences, USA) and eluted with gel-filtration buffer (20 mM Tris, 20 mM NaCl pH 7.5, 1 mM DTT).…”

High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC)

“…To serve rapid cloning, many limitations related to generation of multiple expression plasmids have been recently addressed by high-throughput adaptable systems which enable cloning of hundreds of genes and constructs simultaneously. Several worldwide high-throughput facilities have adopted commercial systems such as Gateway Ò (Life Technologies, USA, CA) [24][25][26] and In-Fusion™ (Clontech, USA, CA) [27], but despite the overall flexibility of these systems, per-reaction cost can be high because of the dependence on proprietary recombinases. Furthermore, the presence of are combination site as part of the open reading frame can affect protein function and solubility.…”