Dual Expression System Suitable for High-Throughput Fluorescence-Based Screening and Production of Soluble Proteins

Abstract

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…MBP fused to either the N-and C-terminus has been shown to increase the expression and folding of eukaryotic fusion proteins expressed in bacteria [27][28][29][30][31]. Although not completely understood [32], fusion of MBP onto a protein has been shown to enhance the solubility of the partner protein [31,[33][34][35]. There are a number of vectors for generating MBP fusion proteins that are commercially available, including the pMAL series from New England Biolabs and pIVEX series from Roche, which contain a specific protease cleavage site in the region between MBP and the multiple cloning site.…”

Delineation of the Xrcc4-interacting Region in the Globular Head Domain of Cernunnos/XLF

“…Purified Cernunnos 1-224 was dialyzed in buffer P (25 mM sodium phosphate, pH 8.0, 150 mM NaCl, 10 mM ␤-mercaptoethanol) and 25 units of benzonase nuclease (Novagen). Human X4 1-203 was subcloned into the pExp vector with a His 6 tag in N-terminal (17). X4 1-203 was expressed in Rosetta (DE3) cells (Novagen) and induced with 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside for 16 h at 20°C.…”

Asymmetric Signal Transduction through Paralogs That Comprise a Genetic Switch for Sugar Sensing in Saccharomyces cerevisiae

“…When fused to a protein of interest, the fusion protein associates with S-protein in a 1:1 ratio, and the degree of this association can be measured by monitoring RNase activity (28), a process enhanced by the use of a fluorophore/quencher, dually labeled RNA substrate (29). Previously, this has been utilized in bacterial systems to quantify protein yield (30); this is the first case of its use in yeast cells.…”