Focus: Expression Cloning
Comparison of the Flexi® Vector Systems to the Gateway® Cloning System
Two expression cloning systems, the Flexi® Vector Systems (Cat.# C8640, C8820, C9320) and the Gateway® Cloning System, were studied to compare their use in a high-throughput environment.
Reprinted from: Blommel, P.G. et al. (2006) Protein Expression and Purification 47, 562–70.
Published in January 2007
Figure 1. Side-by-side comparison of the time spent on the Gateway® and Flexi® Vector cloning protocols. BP reaction refers to generating a Gateway® entry clone from a PCR product. LR reaction refers to transferring the insert from the Gateway® entry to the destination vector.
The success of structural genomics and proteomics initiatives is dependent on the availability of target genes in vectors suitable for protein production. Here, we compare two high-throughput methods for producing expression vectors from plasmid-derived cDNA fragments. Expression vectors were constructed for compatibility with the Gateway® recombination cloning system and the Flexi® Vector restriction-based cloning system. Cloning protocols for each system were conducted in parallel for 96 different target genes from PCR through the production of sequence-verified expression clones. The short nucleotide sequences required to prepare the target open reading frames for Flexi® Vector cloning allowed a single-step PCR protocol, resulting in fewer mutations relative to the Gateway® protocol. Furthermore, through initial cloning of the target open reading frames directly into an expression vector, the Flexi® Vector system gave time and cost savings compared to the protocol required for the Gateway® system (Figure 1). Within the Flexi® Vector system, genes were transferred between four different expression vectors. The efficiency of gene transfer between Flexi® Vectors depended on including a region of sequence identity adjacent to one of the restriction sites. With the proper construction in the flanking sequence of the vector, gene transfer efficiencies of 95–98% were demonstrated.
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