Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.

Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses.

Age, criterion flexibility, and associative recognition.

Objective: The goal of this study was to compare the extent to which young and older adults exhibit flexibility in adjusting decision criteria in response to changes in recognition task difficulty.

Methods: Forty-eight young and 48 older adults studied a list of word pairs and then took 2 successive tests of associative recognition, an easy test consisting of intact study pairs and new lure pairs and a hard test pitting intact study pairs against rearranged lures. The order of the 2 tests was manipulated, with half of the participants in each age group receiving the easy test first and half receiving the hard test first.

Flexi vector cloning.

A protocol for ligation-dependent cloning using the Flexi Vector method in a 96-well format is described. The complete protocol includes PCR amplification of the desired gene to append Flexi Vector cloning sequences, restriction digestion of the PCR products, ligation of the digested PCR products into a similarly digested acceptor vector, transformation and growth of host cells, analysis of the transformed clones, and storage of a sequence-verified clone. The protocol also includes transfer of the sequence-verified clones into another Flexi Vector plasmid backbone.

X-ray structure of a soluble Rieske-type ferredoxin from Mus musculus.

The 2.07 A resolution X-ray crystal structure of a soluble Rieske-type ferredoxin from Mus musculus encoded by the gene Mm.266515 is reported.

High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics.

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC).