Polymorphisms in the selenoprotein S and 15-kDa selenoprotein genes are associated with altered susceptibility to colorectal cancer

Selenium (Se), a dietary trace metal essential for human health, is incorporated into ~25 selenoproteins including selenoprotein S (SelS) and the 15-kDa selenoprotein (Sep15) both of which have functions in the endoplasmic reticulum protein unfolding response. The aim of this study was to investigate whether genetic variants in such selenoprotein genes are associated with altered risk of colorectal cancer (CRC). A Korean population of 827 patients with CRC and 733 healthy controls was genotyped for 7 SNPs in selenoprotein genes and one SNP in the gene encoding manganese superoxide dismutase using Sequenom technology. Multivariate logistic regression analysis showed that after adjustment for lifestyle factors three SNP variants were associated with altered disease risk. There was a mean odds ratio of 2.25 [95% CI 1.13,4.48] in females homozygous TT for rs34713741 in SELS with the T variant being associated with higher risk of rectal cancer, and odds ratios of 2.47 and 2.51, respectively, for rs5845 and rs5859 in SEP15 with the minor A and T alleles being associated with increased risk of male rectal cancer. The data indicate that the minor alleles for rs5845, rs5859 and rs34713741 are associated with increased rectal cancer risk and that the effects of the three SNPs are dependent on gender. The results highlight potential links between Se, the function of two selenoproteins involved in the protein unfolding response and CRC risk. Further studies are required to investigate whether the effects of the variants on CRC risk are also modulated by dietary Se intake

Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product

Background: Filamentous fungi are commonly used as production hosts for bulk enzymes in biotechnological applications. Their robust and quick growth combined with their ability to secrete large amounts of protein directly into the culture medium makes fungi appealing organisms for the generation of novel production systems. The red bread mold Neurospora crassa has long been established as a model system in basic research. It can be very easily genetically manipulated and a wealth of molecular tools and mutants are available. In addition, N. crassa is very fast growing and non-toxic. All of these features point to a high but so far untapped potential of this fungus for biotechnological applications. In this study, we used genetic engineering and bioprocess development in a design-build-test-cycle process to establish N. crassa as a production host for heterologous proteins. Results: The human antibody fragment HT186-D11 was fused to a truncated version of the endogenous enzyme glucoamylase (GLA-1), which served as a carrier protein to achieve secretion into the culture medium. A modular expression cassette was constructed and tested under the control of different promoters. Protease activity was identified as a major limitation of the production strain, and the effects of different mutations causing protease deficiencies were compared. Furthermore, a parallel bioreactor system (1 L) was employed to develop and optimize a production process, including the comparison of different culture media and cultivation parameters. After successful optimization of the production strain and the cultivation conditions an exemplary scale up to a 10 L stirred tank reactor was performed. Conclusions: The data of this study indicate that N. crassa is suited for the production and secretion of heterologous proteins. Controlling expression by the optimized promoter Pccg Inr in a fourfold protease deletion strain resulted in the successful secretion of the heterologous product with estimated yields of 3 mg/L of the fusion protein. The fungus could easily be cultivated in bioreactors and a first scale-up was successful. The system holds therefore much potential, warranting further efforts in optimization