A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the I Ps promoter, an optimized Shine-Dalgamo sequence and the rZ ~I857 repressor. When induced at 42°C such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized.All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated.The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH <2.7 with syringaldazine and ABTS [2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1 and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning.

Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated. The deduced amino acid sequence of PME I is highly similar (74% identity) to the PME from Aspergillus niger. A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme. The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C. The authentic PME I was purified from A. aculeatus culture supernatant and subjected to amino acid sequencing. The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence. Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different. PME I was able to remove 75-85% of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides. When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed. By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate. This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.

HLA-class III region genes may be associated with susceptibility to insulin-dependent diabetes mellitus (IDDM). In this study an NcoI polymorphism of the tumour necrosis factor beta (TNF-beta) gene, which is positioned next to the tumour necrosis factor alpha (TNF-alpha) gene in the HLA class III region, was detected by restriction fragment length polymorphism (RFLP). This polymorphism has previously been reported to be located in the TNF-alpha gene. Caucasian HLA-DR3,4 heterozygous IDDM patients (n = 26) and DR-matched healthy controls (n = 19), as well as randomly selected IDDM patients (n = 27) and controls (n = 25) were studied. In addition four multiplex families (49 individuals) and eight HLA-non-identical sibpairs concordant for IDDM were analysed. The TNF-beta gene RFLP analysis showed fragments of 5.5 kb and 10.5 kb, which behaved as alleles. In all groups there was a haplotype assignment of the TNF-beta 5.5-kb allele to B8,DR3 haplotypes, and of the TNF-beta 10.5-kb allele to B15,DR4-positive haplotypes. The allelic and genotypic frequencies differed between DR3,4 IDDM patients and DR3,4 controls, and the DR3,4 control group differed significantly from the randomly selected control group (P less than 0.0079). In HLA-DR3,4- and DQw8-positive persons, the DR3 haplotypes carried the 10.5-kb allele three times more frequently in IDDM patients than in controls, suggesting that the 10.5-kb allele when present on DR3 haplotypes may contribute to susceptibility to IDDM in DR3,4 heterozygous individuals. A contributory role of the 10.5-kb allele in genetic IDDM susceptibility was supported by the sibpair analysis, in which all were TNF-beta identical. Five were 10.5 kb homozygous, and the remaining three pairs were 5.5/10.5 kb heterozygous. Twenty-five healthy and eight newly diagnosed IDDM patients were randomly selected to study the Escherichia coli lipopolysaccharides (LPS)-purified protein derivate (tuberculin) (PPD)-, and phytohaemagglutinin (PHA)-stimulated monocyte (Mo) secretions of interleukin 1 beta (IL-1 beta) and TNF-alpha in relation to the NcoI TNF-beta gene polymorphism. The LPS- and PHA-stimulated Mo IL-1 beta and TNF-alpha secretions were significantly lower for the TNF-beta 5.5/10.5 kb heterozygous individuals than for TNF-beta 10.5 kb homozygous individuals. Furthermore, the Mo IL-1 beta and TNF-alpha secretions of IDDM patients were significantly higher than the Mo secretions of TNF-beta genotype-matched healthy controls. This study suggests an association between the 10.5 kb TNF-beta allele and IDDM, and demonstrates an association between monokine responses and TNF-beta genotypes.(ABSTRACT TRUNCATED AT 400 WORDS)