Pawel Listwan

Crystal structures of fusion proteins with large-affinity tags

The fusion of a protein of interest to a large‐affinity tag, such as the maltose‐binding protein (MBP), thioredoxin (TRX), or glutathione‐S‐transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large‐affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three‐ to five‐amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.

Keratin Bundling Proteins

Publisher Summary Keratin bundling proteins are a subcategory of intermediate filament associated proteins (IFAPs) that are defined by their ability to organize keratin filaments into macrofibrilar arrays; however, only filaggrin and a related protein have been shown unequivocally to bundle keratin and other intermediate filaments into parallel ropelike structures. This chapter discusses the keratin bundling proteins (particularly the filaggrin—the archetypal keratin bundling protein), filament bundling assays, techniques for the isolation of keratin intermediate filaments, and macrofibril formation assay. Only two proteins—filaggrin and filaggrin-2—have been shown to function as keratin-bundling proteins. It remains to be seen whether this exclusive group will acquire new members, with trichohyalin, hornerin, and repetin all requiring further testing. Although, this chapter focuses on keratin-bundling proteins, it is probable that novel or known IFAPs will be identified that bundle other intermediate filament types such as vimentin and desmin. In addition, the remarkably linear arrays of neurofilaments observed in some axons may be mediated by a specific bundling protein, and the role of neural IFAPs in this activity would seem worthy of further investigation.

Pilot studies on the parallel production of soluble mouse proteins in a bacterial expression system

We investigated the parallel production in medium throughput of mouse proteins, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. The methods were scaled up to allow the simultaneous processing of tens or hundreds of protein samples. Scale-up was achieved in two stages. In an initial study, 30 targets were processed manually but with common protocols for all targets. In the second study, these protocols were applied to 96 target proteins that were processed in an automated manner. The success rates at each stage of the study were similar for both the manual and automated approaches. Overall, 15 of the selected 126 target mouse genes (12%) yielded soluble protein products in a bacterial expression system. This success rate compares favourably with other protein screening projects, particularly for eukaryotic proteins, and could be further improved by modifications at the cloning step.

Overview of the pipeline for structural and functional characterization of macrophage proteins at the university of queensland.

This chapter describes the methodology adopted in a project aimed at structural and functional characterization of proteins that potentially play an important role in mammalian macrophages. The methodology that underpins this project is applicable to both small research groups and larger structural genomics consortia. Gene products with putative roles in macrophage function are identified using gene expression information obtained via DNA microarray technology. Specific targets for structural and functional characterization are then selected based on a set of criteria aimed at maximizing insight into function. The target proteins are cloned using a modification of Gateway cloning technology, expressed with hexa-histidine tags in E. coli, and purified to homogeneity using a combination of affinity and size exclusion chromatography. Purified proteins are finally subjected to crystallization trials and/or NMR-based screening to identify candidates for structure determination. Where crystallography and NMR approaches are unsuccessful, chemical cross-linking is employed to obtain structural information. This resulting structural information is used to guide cell biology experiments to further investigate the cellular and molecular function of the targets in macrophage biology. Jointly, the data sheds light on the molecular and cellular functions of macrophage proteins.