Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin.

The extracellular ligand-binding domain (EPObp) of the human EPO receptor (EPOR) was expressed both in CHO (Chinese Hamster Ovary) cells and in Pichia pastoris. The CHO and yeast expressed receptors showed identical affinity for EPO binding. Expression levels in P. pastoris were significantly higher, favoring its use as an expression and scale-up production system. Incubation of EPO with a fourfold molar excess of receptor at high protein concentrations yielded stable EPO-EPObp complexes. Quantification of EPO and EPObp in the complex yielded a molar ratio of one EPO molecule to two receptor molecules. Residues that are responsible for EPOR glycosylation and isomerization in Pichia were identified and eliminated by site-specific mutagenesis. A thiol modification was identified and a method was developed to remove the modified species from EPObp. EPObp was complexed with erythropoietin (EPO) and purified. The complex crystallized in two crystal forms that diffracted to 2.8 and 1.9 A respectively. (Form 1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals, Inc. and Amgen, Inc. respectively.) Both contained one complex per asymmetric unit with a stoichiometry of two EPObps to one EPO.

Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation.

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant alpha-synuclein form insoluble fibrillar aggregates with antiparallel beta-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of alpha-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.

Efficiency of signalling through cytokine receptors depends critically on receptor orientation.

Human erythropoietin is a haematopoietic cytokine required for the differentiation and proliferation of precursor cells into red blood cells. It activates cells by binding and orientating two cell-surface erythropoietin receptors (EPORs) which trigger an intracellular phosphorylation cascade. The half-maximal response in a cellular proliferation assay is evoked at an erythropoietin concentration of 10 pM, 10(-2) of its Kd value for erythropoietin-EPOR binding site 1 (Kd approximately equal to nM), and 10(-5) of the Kd for erythropoietin-EPOR binding site 2 (Kd approximately equal to 1 microM). Overall half-maximal binding (IC50) of cell-surface receptors is produced with approximately 0.18 nM erythropoietin, indicating that only approximately 6% of the receptors would be bound in the presence of 10 pM erythropoietin. Other effective erythropoietin-mimetic ligands that dimerize receptors can evoke the same cellular responses but much less efficiently, requiring concentrations close to their Kd values (approximately 0.1 microM). The crystal structure of erythropoietin complexed to the extracellular ligand-binding domains of the erythropoietin receptor, determined at 1.9 A from two crystal forms, shows that erythropoietin imposes a unique 120 degrees angular relationship and orientation that is responsible for optimal signalling through intracellular kinase pathways.

Interactions between NFkappaB and its inhibitor ikappaB: biophysical characterization of a NFkappaB/ikappaB-alpha complex.

The N-terminal domain (1-318 amino acids) of mouse NFkappaB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length ikappaB-alpha (MAD3, 1-317 amino acids) molecule was generated by binding the E. coli-derived ikappaB-alpha to the purified NFkappaB and purifying the complex by sequential chromatography. The stoichiometry of NFkappaB to ikappaB in the complex was determined to be 2 to 1 by light scattering and SDS-polyacrylamide gel electrophoresis. The secondary structure of the NFkappaB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFkappaB occurs upon binding of DNA. The FTIR spectrum of the NFkappaB/ikappaB complex indicates that its secondary structure is composed of 17% alpha-helix, 39% beta-strand, 18% irregular structures, and 26% beta-turns and loops. By comparing these data to the FTIR data for NFkappaB alone, it is concluded that the ikappaB (MAD3) in the complex contains 35% alpha-helix, 27% beta-strand, 22% irregular structures, and 16% beta-turns and loops. Circular dichroism (CD) analysis of a shorter form of ikappaB (pp40) indicates that it contains at least 20% alpha-helix and that the ikappaB subunit accounts for nearly all of the alpha-helix present in the NFkappaB/ikappaB complex, consistent with the FTIR results. The stabilities of NFkappaB, ikappaB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of ikappaB is enhanced upon the formation of the NFkappaB/ikappaB complex.

Use of a modified tryptophanase promoter to direct high-level expression of foreign proteins in E. coli.

We have modified the tryptophanase promoter (PtnaA) for use as a temperature-independent promoter for the production of recombinant proteins. Although any protein will have a temperature range in which its expression is optimal, we find the tryptophanase promoter functions at all physiologically relevant temperatures (20 degrees C to 42 degrees C). Induction at temperatures below 37 degrees C avoids eliciting the heat-shock response and may favor the production of protein in the soluble state. A short segment of the E. coli tnaA promoter containing the catabolite gene activator protein (CAP) binding site but no tryptophan-responsive elements was used to direct synthesis of various proteins. Conditions for high cell density fermentation and induction control were developed. Expression was induced by depletion of glucose and was maximal when an alternative nonrepressing carbon source was supplied. Expression of certain proteins was tightly controlled; however, pre-induction expression was observed with other reporter genes. The tnaC leader portion of the tnaA promoter was found to reduce pre-induction expression in the presence of glucose, although maximal expression was observed only in the absence of this region. The effect of temperature on expression of several recombinant proteins was investigated. Although some proteins were produced only in inclusion bodies as insoluble material, the production of one protein in soluble form was clearly temperature dependent.

Use of a ring chromosome and pulsed-field gels to study interhomolog recombination, double-strand DNA breaks and sister-chromatid exchange in yeast.

We describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome III (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. We demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints, we present data on the timing of commitment to meiotic recombination scored genetically. We have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-sized circles originating in part from sister-chromatid exchange, which we find to be frequent during meiosis.

Purification of DNA polymerase II, a distinct DNA polymerase, from Saccharomyces cerevisiae.

Yeast DNA polymerases I and III have been well characterized physically, biochemically, genetically and immunologically. DNA polymerase II is present in very small amounts, and only partially purified preparations have been available for characterization, making comparison with DNA polymerases I and III difficult. Recently, we have shown that DNA polymerases II and III are genetically distinct (Sitney et al., 1989). In this work, we show that polymerase II is also genetically distinct from polymerase I, since polymerase II can be purified in equal amounts from wild-type and mutant strains completely lacking DNA polymerase I activity. Thus, yeast contains three major nuclear DNA polymerases. The core catalytic subunit of DNA polymerase II was purified to near homogeneity using a reconstitution assay. Two factors that stimulate the core polymerase were identified and used to monitor activity during purification and analysis. The predominant species of the most highly purified preparation of polymerase II is 132,000 Da. However, polymerase activity gels suggest that the 132,000-Da form of DNA polymerase II is probably an active proteolytic fragment derived from a 170,000-Da protein. The highly purified polymerase fractions contain a 3'----5'-exonuclease activity that purifies at a constant ratio with polymerase during the final two purification steps. However, DNA polymerase II does not copurify with a DNA primase activity.

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene.

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.

First position wobble in codon-anticodon pairing: amber suppression by a yeast glutamine tRNA.

A 2.4-kb fragment of DNA isolated from the Saccharomyces cerevisiae genome was found to suppress amber mutations when its carrier plasmid was present in high copy number. A 1.2-kb subclone of this fragment was sufficient to confer suppressor activity. Sequencing has established that this fragment carries a normal glutamine tRNA gene. Deletion of this tRNA gene from the subclone resulted in the loss of suppressor activity. The tRNAGln has the anticodon CUG that normally recognizes the glutamine codon CAG. We propose that suppression occurs via an inefficient readthrough of the UAG amber stop codons during translation. Such readthrough requires wobble in the first position of the codon.