A hemofiltration system for a mammal comprises a hemofilter and an adsorbent device. The hemofilter removes ultrafiltrate from a blood stream extracted from the mammal to create a filtered blood stream and an ultrafiltrate stream. The adsorbent device is comprised of one or more adsorbent materials and is used to adsorb inflammatory mediators from the ultrafiltrate stream received from the hemofilter removing inflammatory mediators that cause inflammatory mediator related disease, sepsis, and SIRS/MODS/MOSF to create a post adsorption ultrafiltrate stream. The post adsorption ultrafiltrate stream is selectively combined with the filtered blood stream and together with the filtered blood stream is returned to the mammal.

Hemofiltration systems, methods and devices used to treat inflammatory mediator related disease

The extent to which the order of genes along chromosomes is conserved between Saccharomyces cerevisiae and related species was studied by analysing data from DNA sequence databases. As expected, the extent of gene order conservation decreases with increasing evolutionary distance. About 59% of adjacent gene pairs in Kluyveromyces lactis or K. marxianus are also adjacent in S. cerevisiae, and a further 16% of Kluyveromyces neighbours can be explained in terms of the inferred ancestral gene order in Saccharomyces prior to the occurrence of an ancient whole-genome duplication. Only 13% of Candida albicans linkages, and no Schizosaccharomyces pombe linkages, are conserved. Analysis of gene order arrangements, chromosome numbers, and ribosomal RNA sequences suggests that genome duplication occurred before the divergence of the four species in Saccharomyces sensu stricto (all of which have 16 chromosomes), but after this lineage had diverged from Saccharomyces kluyveri and the Kluyveromyces lactis/marxianus species assemblage. © 1998 John Wiley & Sons, Ltd.

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Evolution of gene order and chromosome number in Saccharomyces, Kluyveromyces and related fungi.

The unfolded protein response (UPR) activates Ire1, an endoplasmic reticulum (ER) resident transmembrane kinase and ribonuclease (RNase), in response to ER stress. We used an in vivo assay, in which disappearance of the UPR-induced spliced HAC1 messenger ribonucleic acid (mRNA) correlates with the recovery of the ER protein-folding capacity, to investigate the attenuation of the UPR in yeast. We find that, once activated, spliced HAC1 mRNA is sustained in cells expressing Ire1 carrying phosphomimetic mutations within the kinase activation loop, suggesting that dephosphorylation of Ire1 is an important step in RNase deactivation. Additionally, spliced HAC1 mRNA is also sustained after UPR induction in cells expressing Ire1 with mutations in the conserved DFG kinase motif (D828A) or a conserved residue (F842) within the activation loop. The importance of proper Ire1 RNase attenuation is demonstrated by the inability of cells expressing Ire1-D828A to grow under ER stress. We propose that the activity of the Ire1 kinase domain plays a role in attenuating its RNase activity when ER function is recovered.

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Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase.

Endostatin is a potent angiogenesis inhibitor. In order to isolate sufficient quantities of soluble protein for in vivo studies in mice, we expressed murine endostatin in Pichia pastoris. Analysis of the expressed protein by mass spectrometry indicated that the protein was truncated. N-terminal sequence analysis determined that the N-terminus was intact, suggesting that the C-terminal lysine was missing. In Saccharomyces cerevisiae, Kex1p can cleave lysine and arginine residues from the C-terminus of peptides and proteins. We hypothesized that the KEX1 homologue in P. pastoris is responsible for the loss of the C-terminal lysine of endostatin. To test this hypothesis, we cloned and disrupted the P. pastoris KEX1 gene. Although the overall amino acid identity between the P. pastoris and the S. cerevisae Kex1p is only 36%, the amino acid residues involved in the catalytic activity or close to the active residues are highly conserved. Disruption of the KEX1 reading frame allowed expression of murine and human endostatin with the C-terminal lysine. The KEX1 disruption strain may be a useful tool for the expression of other proteins with a C-terminal basic amino acid. Addition of a lysine to the C-terminus of recombinant proteins may protect the C-terminus from degradation by other carboxypeptidases.

Disruption of the KEX1 gene in Pichia pastoris allows expression of full-length murine and human endostatin.

A process for the preparation of albumin which has extremely low levels of or is essentially free of colorants, metal ions, human proteins, fragments of albumin, polymers or aggregates of albumin, and viruses, and which is relatively non-glycated, relatively high in free thiol and with an intact C-terminus. The process comprises passing albumin (preferably expressed and secreted by transformed yeast) through two chromatography purifications, ultrafiltering the product, passing through two further chromatography steps and again ultrafiltering the product.

High purity albumin and method of producing

Human serum albumin obtained by gene manipulation techniques can be purified by a combination of specified steps in which a culture supernatant obtained from a human serum albumin-producing host is subjected to ultrafiltration, heat treatment, acid treatment and another ultrafiltration, followed by subsequent treatments with a cation exchanger, a hydrophobic chromatography carrier and an anion exchanger, and by salting-out to thereby obtain a pure form of human serum albumin which contains substantially no proteinous and polysaccharide contaminants, which is formulated into a pharmaceutical preparation. The thus obtained human serum albumin can further be purified by treating recombinant human serum albumin with a hydrophobic chromatography carrier at pH of 2 to 5 and a salt concentration of 0.4 to 1 and exposing the carrier to a pH of 6 to 8 and a salt concentration of 0.01 to 0.3 M, or treating the culture supernatant with boric acid or a salt thereof at pH 8 to 11 for 1 to 10 hours and recovering the supernatant. This process makes it possible to effeciently purify recombinant human serum albumin and to provide substantially pure human serum albumin which does not contain producer host-related substances and other contaminants and is sufficiently free from coloration.

Recombinant human serum albumin, process for producing the same and pharmaceutical preparation containing the same

We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH2-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene Using the S cerevisiae PRC1 gene as a hybridization probe, a cross-hybridizing fragment of P pastoris genomic DNA was identified and the gene, PRC1, encoding CPY, was cloned The open reading frame of the P pastoris PRC1 gene consists of 1569 bp encoding a protein of 523 amino acids The molecular mass of the protein is calculated to be 5944 kDa without sugar chains The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites The NH2-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant There is 61% identity between the amino acid sequences of P pastoris Prc1p and S cerevisiae Prc1p Chromosomal disruption of the PRC1 gene resulted in the loss of CPY activity Over-expression of the PRC1 gene under regulation of the P pastoris AOX1 promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY The nucleotide sequence data reported in this paper will appear in the EMBL Nucleotide Sequence Databases under the Accession Number X87987

Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y.

PURPOSE:To obtain an almost colorless human serum albumin free from contaminant components by purifying cultured supernatant of a human serum albumin- producing host by ultrafiltration, heating, acid treatment, cation and anion exchange treatment, hydrophobic chromatography, etc CONSTITUTION:Cultured supernatant of a human serum albumin-producing host is filtered with an ultrafiltration membrane having a fractionation molecular weight of 100,000-500,000 and 1,000-50,000 and the filtrate is heat-treated at 50-70 degC for 30min to 5hr The heat-treated product is treated with an acid at pH3-5, filtered with an ultrafiltration membrane having a fractionation molecular weight of 100,000-500,000, subjected to cation exchange at pH3-5 and a salt concentration of 001-02M, eluted at pH8-10 and a salt concentration of 02-05M, subjected to hydrophobic chromatography at pH6-8 and a salt concentration of 001-05M to collect the non-adsorbed fraction and subjected to anion exchange treatment at pH6-8 and a salt concentration of 001-01M to obtain the objective purified human serum albumin from the non-adsorbed fraction

Production of human serum albumin obtained by genetic engineering and composition containing human serum albumin produced thereby

A method for decoloring a recombinant human serum albumin by treating the albumin with a reducing agent is disclosed. Also, a method for decoloring a recombinant human serum albumin by treating the albumin with a method removing free polysaccharides with a cation exchanger followed by heat treatment is disclosed. The present invention provides a recombinant human serum albumin, coloring of which is fully suppressed by preventing binding of certain coloring components, which are contained in the raw materials or contaminants secreted by a microorganism, to human serum albumin so as not to cause coloring of the human serum albumin.

Method for decoloring human serum albumin

PROBLEM TO BE SOLVED: To obtain an inexpensive human serum albumin preparation, capable of stably feeding collecting source, free from danger of mixing of virus, etc., and suitable for medical uses by purifying human serum albumin derived from generic engineering by combination of specific steps. SOLUTION: This human serum albumin-containing composition is obtained purifying human serum albumin derived from generic engineering and contains no producing host-related ingredients or other impurity ingredients and sufficiently suppressed in coloring. The composition is preferably obtained by carrying out ultrafiltration, heat treatment and acid treatment of cultured supernatant of a host producing human serum albumin and carrying out ultrafiltration of the treated material and subjecting the treated material to each treatment due to cation exchanger, hydrophobic chromatography, anionic exchanger, salting out, etc.

Naoto Furuhata

Papers

Recombinant human serum albumin, process for producing the same and pharmaceutical preparation containing the same

Cloning and characterization of the Pichia pastoris PRC1 gene encoding carboxypeptidase Y.

Production of human serum albumin obtained by genetic engineering and composition containing human serum albumin produced thereby

Method for decoloring human serum albumin

High-purity human serum albumin-containing composition obtained by human serum albumin derived from genetic engineering