Foreign gene expression in yeast: a review.

The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disorders or conditions using albumin fusion proteins of the invention.

Albumin Fusion Proteins

Extension of Recombinant Human RANTES by the Retention of the Initiating Methionine Produces a Potent Antagonist

https://epublications.marquette.edu/cgi/viewcontent.cgi?article=1330&context=chem_fac

The proteomics of N-terminal methionine cleavage.

The methylotrophic industrial yeast, Pi–chia pastoris, has been developed as a host system for the large–scale production of heterologous proteins. As an example of the systems, the synthesis of hepatitis B surface antigen (HBsAg) is described. Expression of the HBsAg gene is controlled by a methanol–regulated promoter derived from the primary alcohol oxidase gene, AOX1, of P. pastoris. The highest level of HBsAg is observed in a novel mutant strain of P. pastoris in which the AOX1 structural gene is deleted and replaced by a construction composed of the AOX1 promoter–HBsAg gene expression cassette. When grown on methanol, 2–3% of the soluble protein in this strain is HBsAg. Characterization of the recombinant HBsAg indicates that virtually all of the P. pastoris–synthesized HBsAg exists in a form which is similar to human serum–derived HBsAg 22 nm particles. Scale–up of the HBsAg production process from shake–flask to high cell–density fermentor cultures is described as well. A single culture of the P. pastoris HBsAg expression strain in a volume of 240 liters and at a density of 60 grams/liter dry weight of cells produced 0.4 grams/liter, or a total of 90 grams of HBsAg 22 nm particles, an amount sufficient for approximately 9 million doses of vaccine.

High–Level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast, Pichia Pastoris

Hirudin synthesized by the medicinal leech Hirudo medicinalis is the most potent antithrombic factor found in nature. We have used Saccharomyces cerevisiae for the efficient production of recombinant hirudin variant 2 (rHV2). The mature hirudin coding sequence was placed in frame with the prepro sequence of the yeast MFα1 gene. Multicopy plasmids carrying different fused genes transcribed from the MFα1 promoter were used to transform a yeast strain selecting for uracil prototrophy. Analysis of proteins from culture supernatants revealed efficient secretion of hirudin in all cases. However, only precursors in which the HV2 mature sequence directly followed the cleavage site for yscF (a protease involved in the maturation of yeast α factor) were processed correctly to yield biologically active hirudin.

Expression and Secretion in S. Cerevisiae of Biologically Active Leech Hirudin

Simple auxotrophic mutants of S. cerevisiae transformed to prototrophy via plasmid DNA must be grown in media in which cured cells are counter–selected. This restricts the choice of the medium to those whose composition is compatible with the selection. These media are generally not comparable with the cheap, complex media used by industry for the production of yeast cells. We report a new selection system that enables the growth of plasmid–transformed yeast cells for the production of foreign proteins in complex media. The recipient strains are double–mutants (ura3 fur1) whose viability is strictly linked to the presence of a plasmid encoding a functional orotidine–5′–phosphate decarboxylase (OMP decase). We show that these strains can produce high levels of a plasmid–encoded foreign protein (namely human α1–antitrypsin) in various complex media, including those used by industry, for many generations without any detectable loss of the plasmid–linked phenotype.

Plasmid–Transformed ura3 fur1 Double-Mutants of S. cerevisiae : An Autoselection System Applicable to the Production of Foreign Proteins

Functional block of DNA enabling to prepare hirudine from yeast, characterized in that it comprises at least: - the gene of hirudine or one of its variants (gene H); - A DNA sequence (Str) comprising the signals providing for the transcription of the gene H by the yeast.

Expression and secretion vectors for hirudine by way of transformed yeasts

Double mutants of Saccharomyces cerevisiae harbour stable plasmids: stable expression of a eukaryotic gene and the influence of host physiology during continuous culture

Hirudine variants comprising an amino acid other than the amino acid of the natural form in position 47 or 63.

Gerard Loison

Papers

Expression and Secretion in S. Cerevisiae of Biologically Active Leech Hirudin

Plasmid–Transformed ura3 fur1 Double-Mutants of S. cerevisiae : An Autoselection System Applicable to the Production of Foreign Proteins

Expression and secretion vectors for hirudine by way of transformed yeasts

Double mutants of Saccharomyces cerevisiae harbour stable plasmids: stable expression of a eukaryotic gene and the influence of host physiology during continuous culture

Hirudine variants, their use and preparation