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Biomedical subjects

U Weydemann

Publications and source records attributed to U Weydemann.

6 recordsLinked to original sources
PubMed1995

High-level secretion of hirudin by Hansenula polymorpha--authentic processing of three different preprohirudins.

A DNA sequence coding for a subtype of the hirudin variant HV1 was expressed in the methylotrophic yeast Hansenula polymorpha from a strongly inducible promoter element derived from a gene of the methanol metabolism pathway. For secretion, the coding sequence was fused to the KEX2 recognition site of three different prepro segments engineered from the MF alpha 1 gene of Saccharomyces cerevisiae, the glucoamylase (GAM1) gene of Schwanniomyces occidentalis and the gene for a crustacean hyperglycemic hormone from the shore crab Carcinus maenas. In all three cases, correct processing of the precursor molecule and efficient secretion of the mature protein were observed. In fermentations on a 10-1 scale of a transformant strain harbouring a MF alpha 1/hirudin-gene fusion yields in the range of grams per litre could be obtained. The majority of the secreted product was identified as the full-length 65-amino-acid hirudin. Only small amounts of a truncated 63-amino- acid product, frequently observed in S. cerevisiae-based expression systems, could be detected.

Amino Acid Sequence↗
PubMed1995

Stable multicopy integration of vector sequences in Hansenula polymorpha.

Plasmids without an origin of replication, but bearing the URA3 gene of Saccharomyces cerevisiae as a selective marker for transformation, are shown to replicate autonomously in Hansenula polymorpha, indicating that parts of the S. cerevisiae URA3 gene can fulfil an autonomous replication and stabilization function in H. polymorpha. Such plasmids, replicated in low copy number in monomeric conformation, could be rescued in E. coli, and showed a low mitotic stability under selective and non-selective conditions. Selective propagation of such transformants, however, led to the integration of plasmid sequences into the H. polymorpha genome. The integration event usually occurred in high copy number (approx. 30-50) at a single non-homologous site of the genome. The plasmid sequences were found to be present in tandem array and stable under non-selective conditions. In contrast, the use of homologous URA3 gene under similar conditions led to low-copy-number transformants.

DNA Replication↗
PubMed1992

Heterologous protein production in yeast.

The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeast Saccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application. In the following review a selection from the different yeast systems is described and compared.

Gene Expression↗
PubMed1991

The duplicated chalcone synthase genes C2 and Whp (white pollen) of Zea mays are independently regulated; evidence for translational control of Whp expression by the anthocyanin intensifying gene in.

Two chalcone synthase genes in maize have been cloned and molecularly characterized to be the C2 and the Whp (white pollen) locus. The two genes have highly homologous exon sequences but differ considerably in sequences 5' upstream and 3' downstream of the coding region, as well as in their introns. Northern and Western experiments of chalcone synthase expression in various tissues and in different genotypes indicated that C2 and Whp are differently regulated. The expression of Whp in maize aleurone is dependent on the presence of the recessive allele of the gene intensifier (in). The regulatory effect of in on Whp expression is not detectable at the transcriptional level, but seems to take place during translation.

Acyltransferases↗
PubMed1991

Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase.

We have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein. Transformants of H. polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified. Yields of up to 1.4g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter.

Amino Acid Sequence↗