A Versatile and Efficient High-Throughput Cloning Tool for Structural Biology

Abstract

Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.

Molecular basis for inhibition of AcrB multidrug efflux pump by novel and powerful pyranopyridine derivatives

“…The gene coding for AcrBper was created by amplifying the acrB segments coding for A39-T329 and S561-S869 with primers that connect the two regions via a GGSGGSGGS linker upon BamHI digestion and ligation (30). AcrBper was expressed in E. coli MC1061 (30) and was purified by using its C-terminal His tag sequence.…”

Bicistronic mRNAs to Enhance Membrane Protein Overexpression

“…Though the fluorescence of C-terminal GFP fusion proteins is an indirect indication for correct folding, a very high correlation between GFP fluorescence and functional and stably folded membrane proteins is generally observed [5,6,32]. ORFs coding for the glycine betaine/proline ABC transporter permease ProW [5,33], the mechanosensitive channel of large conductance MscL [33,34], the lactose permease LacY [6,35] and the glutamate transporter GltP [6] were placed under the control of the arabinose promoter [36] and fused to the sequence coding for a C-terminal GFP in a diverse set of universal high-throughput cloning vectors ( Fig.1) [37]. These vectors allowed direct expression of the target ORF-GFP alone, with translational fusions to Mistic, SUMO or YbeL, or with transcriptional fusions to mstX, sumo, or ybeL (Fig.…”

“…To enable the insertion of various sequences preceding the target ORFs, vector pBXC3GH [37] was modified to contain a unique XhoI site between the start codon and the serine codon that immediately preceded the target ORF. The XhoI site was introduced by PCR using phosphorylated primers.…”

Unusual Structural Features Revealed by the Solution NMR Structure of the NLRC5 Caspase Recruitment Domain

“…As a result, a glutathione S-transferase (GST) tag, together with a human rhinovirus 3C protease (GE healthcare) cleavage site was added to the N-terminus of the mouse NLRC5 atypical CARD. Mouse RIG-I tandem CARD (residues 1 to 186) was cloned into a modified pBAD24 expression vector with FX cloning 20 , adding a human rhinovirus 3C protease (GE healthcare) cleavage site, a eGFP-tag, and decahistidine affinity tag to the C-terminus. Transformed…”

“…NLRC5 and RIG-I plasmid constructs included mouse NLRC5, encoding residues 1-96 and 1-130 and denoted as mu96 and mu130, respectively; human NLRC5, encoding residues 1-96, 1-130, 1-550, and 1-1866 denoted as hu96, hu130, hu550, hu1866, and hu1866-eGFP, respectively; mouse RIG-I, encoding residues 1-186, marked as muRIG-I 186; and human RIG-I, encoding residues 1-186 and 1-925, marked as huRIG-I 186 and huRIG-I 925, respectively. All molecular cloning was performed with standard PCR methods in combination with FX cloning 20 .…”