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Citations Search

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Methods in Mol. Biol. 577, 25-39. High-Throughput Construction of ORF Clones for Production of the Recombinant Proteins 2009

Yamakawa, Hisashi

Notes: The authors use the Flexi® Cloning System to convert their cDNA clones to expression-ready clones. They wanted clones that could be used for comprehensive analysis with the HaloTag® Technology. They also describe a method of transferring ORFs between Flexi® Vectors in a 96-well plate format. They also used Wizard® SV 96 Plasmid DNA Purification, Wizard® SV PCR Clean-Up, and Wizard® SV Gel and PCR Clean-Up Systems. (4056)

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DNA Research 15, 137-149. Exploration of human ORFeome: High-throughput preparation of ORF clones and efficient characterization of their protein products. 2008

Nagase, T., Yamakawa, H., Tadokoro, S., Nakajima, D., Inoue, S., Yamaguchi, K., Itokawa, Y., Kikuno, R.F., Koga, H. and Ohara, O.

Notes: These authors used the Flexi® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi® System and Gateway® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

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J. Proteome Res. 7(10), 4475-82. Improving protein array performance: focus on washing and storage conditions. 2008

Nath, N., Hurst, R., Hook, B., Meisenheimer, P., Zhao, K.Q., Nassif, N., Bulleit, R.F., and Storts, D.R.

Notes: These authors tested the effect of different washing, drying and storage conditions on the stability of various protein:protein interaction arrays. They tested five different interacting protein pairs and three enzymes, monitoring stability and activity under various processing conditions. They found that addition of 5% glycerol to the wash buffer helped retain enzyme activity during washing and drying. There was significant loss of enzyme activity when slides were stored dry at 4oC but slides stored at -20oC in 50% glycerol retained enzyme activity. HaloTag-fused enzymes in cell-free extracts could undergo multiple freeze-thaw cycles without significant effect on enzyme activity, indicating that such extracts could be frozen at -70oC and used to print small batches of arrays at various intervals over a period of weeks. (3890)

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Protein Expr. Purif. 47, 562–570. High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system. 2006

Blommel, P.G., Martin, P.A., Wrobel, R.L., Steffen, E. and Fox, B.G.

Notes: In this study, the Flexi® Vector Systems was compared with the Gateway® Cloning System to determine its utility in high-throughput expression cloning by subcloning 96 human target genes. A direct comparison between pVP16, the Gateway® vector and the equivalent Flexi® Vector, pVP33A or K, was achieved by modifying pVP16 with the barnase gene and PmeI/SgfI restriction sites, duplicating the design available in the commercial Flexi® Vectors. Capture of genes by PCR amplification of the cDNAs was similar for both systems, but the timeline for the Flexi® Vector system was shorter at 6–8 days compared to 12 days for the Gateway® system. They also found the Flexi® Vector System was lower cost and more accurate due to the shorter primers required for the Flexi® Vector cloning. The authors found nearly twofold fewer missense errors due to the shorter amplification primers. Ninty-six cDNAs were amplified simultaneously in their protocol and PCR products were cleaned up using either the MagneSil® PCR Clean-Up System or Wizard® SV 96 PCR Clean-Up, ligated into the Flexi® Vector, and transformed into Select96™ Competent Cells. The study also compared transfer of cDNA inserts between different Flexi® Vectors and transfer of cDNA inserts between different Gateway® vectors and found similar performance in the two systems. For the Flexi® Vector test set, the authors sequenced the clones, validating the high fidelity transfer of cDNA inserts between Flexi® Vectors. (3533)

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