Tubular reabsorption rates for myoglobin in the isolated perfused rat kidney.

The renal handling of myoglobin has been studied in the isolated perfused rat kidney. Myoglobin was freely filtered. Reabsorption by the renal tubules showed saturation kinetics with a relatively low maximum rate of reabsorption (Tmax) of 27-30 micrograms min-1 g-1 wet wt. at a perfusate concentration of 70-80 micrograms/ml. Myoglobin reabsorption is therefore much less than that reported for immunoglobulin light chain or lysozyme in this model. Large increases in sodium and water excretion produced by omission of oncotic agent from the perfusate did not alter the kinetics of myoglobin reabsorption. The use of bovine serum albumin as oncotic agent in the perfusate prevented the tubular reabsorption of myoglobin. Small amounts of albumin are filtered by the isolated perfused kidney and it is postulated that this albumin interferes with tubular reabsorption of myoglobin

A novel method for the purification of HIV-1 p24 protein from hybrid Ty virus-like particles (Ty-VLPs).

The self-assembly properties of a protein encoded by the yeast retrotransposon Ty can be exploited to produce large amounts of recombinant, particulate fusion proteins as hybrid Ty virus-like particles (Ty-VLPs). This system has now been adapted to allow the release of the additional protein by incorporation of a protease cleavage site between the yeast carrier protein and the protein of interest. The purification of the additional protein is facilitated by exploiting the ease with which Ty-VLPs can be purified from other yeast cell components due to their particulate nature. We have used this modified system to produce hybrid particles containing the HIV-1 p24 protein downstream of the recognition sequence for the blood coagulation factor Xa. The p24 was released from the particles by proteolytic cleavage and rapidly separated from the residual particulate material using centrifugation and standard chromatography techniques. This procedure has been used to purify milligram quantities of HIV-1 p24 protein that reacts with anti-p24 sera and elicits the production of p24-specific antibodies in experimental animals

Identification of the Minimal Conserved Structure of the HIV-Reverse Transcriptase under the Presence and Absence of Drug Pressure

Identification of the Minimal Conserved Structure of the HIV-Reverse Transcriptase under the Presence and Absence of Drug Pressur

Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs