Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in E. coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible.Here we use a quantitative, high throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove 4 of the 5 arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 °C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of ~400 mg/ L of expression culture (~15 mg pure TEV protease per g of E. coli cell paste). Methods for producing glutathione S-transferase tagged TEV with similar yields (~12 mg pure protease fusion per g of E. coli cell paste) are also reported.

The auto-induction method of protein expression in E. coli is based on diauxic growth resulting from dynamic function of lac operon regulatory elements (lacO and LacI) in mixtures of glucose, glycerol and lactose. The results show that successful execution of auto-induction is strongly dependent on the plasmid promoter and repressor construction, on the oxygenation state of the culture, and on the composition of the auto-induction medium. Thus expression hosts expressing high levels of LacI during aerobic growth exhibit reduced ability to effectively complete the auto-induction process. Manipulation of the promoter to decrease the expression of LacI altered the preference for lactose consumption in a manner that led to increased protein expression and partially relieved the sensitivity of the auto-induction process to the oxygenation state of the culture. Factorial design methods were used to optimize the chemically defined growth medium used for expression of two model proteins, Photinus luciferase and enhanced green fluorescent protein, including variations for production of both unlabeled and selenomethionine-labeled samples. The optimization included studies of the expression from T7 and T7-lacI promoter plasmids and from T5 phage promoter plasmids expressing two levels of LacI. Upon the basis of the analysis of over 500 independent expression results, combinations of optimized expression media and expression plasmids that gave protein yields of greater than 1000 μg/mL of expression culture were identified.

This unit contains protocols for the use of lactose-derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X-ray structural studies, and multi-well plate growth for screening and optimization are also included.

The Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsis open reading frames into Escherichia coli expression vectors are presented along with an analysis of results from approximately 2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold-function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel Gateway protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the Gateway system.

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