Mogens Christensen

Rhizomucor miehei triglyceride lipase is synthesized as a precursor.

ARhizomucor miehie cDNA library constructed inEscherichia coli was screened with synthetic oligonucleotides designed from knowledge of a partial amino acid sequence of the secreted triglyceride lipase (triacyl-glycerol acylhydrolase EC 3.1.1.3) from this fungus. Lipase-specific recombinants were isolated and their insert sequenced. Unlike characterized bacterial and mammalian triglyceride lipases, the fungal enzyme is synthesized as a precursor, including a 70 amino acid residue propeptide between the 24 amino acid residues of the signal peptide and the 269 residues of the mature enzyme. The precursor processing mechanism, which involves cleavage between a methionine and a serine residue, is unknown. By sequence comparison with other lipases, a serine residue involved in substrate binding was identified in the fungal lipase. The sequence around this residue is well-conserved among characterized lipases. Conservation of an intron in an isolated cDNA recombinant and immunoprecipitation of in vitro synthesizedR. miehei translation products indicates that the expression of the lipase gene might involve inefficient mRNA splicing.

Rhizomucor miehei triglyceride lipase is processed and secreted from transformedAspergillus oryzae

The cDNA encoding the precursor of theRhizomucor miehei triglyceride lipase was inserted in anAspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of theAspergillus oryzae α-amylase gene promoter and theAspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced intoAspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted fromA. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the nativeRhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted fromAspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

Purification and characterization of the trefoil peptide human spasmolytic polypeptide (hSP) produced in yeast

Recombinant human spasmolytic polypeptide (r‐hSP) has been produced in relatively large amounts in Saccharomyces cerevisiae. The two intronless trefoil domains of the hSP‐DNA were cloned separately by PCR from human genomic DNA, and the remaining parts of the gene synthezised. Recombinant plasmids were constructed to encode a fusion protein consisting of a hybrid leader sequence and the hSP sequence. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Kex 2 processing enzyme system. The secreted r‐hSP was found in a glycosylated and an non‐glycosylated form. The two forms of r‐hSP were purified from the yeast fermentation broth by a combination of ion‐exchange chromatography and preparative HPLC. The overall yield from 8 litres of fermentation broth was 160 mg r‐hSP and 219 mg glycosylated r‐hSP corresponding to 50% and 34%, respectively. The structure of the r‐hSP and the glycosylated r‐hSP was determined by amino acid analysis and carbohydrate composition analysis as well as by peptide mapping, amino acid sequencing and mass spectrometric analysis.

Generation of Gla-domainless FVIIa by cathepsin G-mediated cleavage.

Cougulation factor VII contains ten λ‐carboxyglutamic acid residues in the N‐terminal region (Gla‐domain) which are essential for the hemostatic function of FVII. The present study shows that granulocyte cathepsin G degrades the Gla‐domain of FVIIa in vitro. Characterization of the truncated FVIIa by SDS‐PAGE and N‐terminal amino acid sequence analysis revealed that cleavage had occurred between Tyr‐44 and Ser‐45 and that further cleavage was only obtained on extensive cathepsin G exposure. Cleavage of vitamin K‐dependent coagulation factors by cathepsin G may play a role in vivo, and it offers a convenient way of obtaining proteins deprived of their Gla‐domain for functional and structural studies.