Andrew P. Wilkinson

Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme

The cDNA gene encoding porcine pancreatic prophospholipase A2 (proPLA2) was cloned into an Aspergillus niger expression vector downstream of the glucoamylase (glaA) gene promoter region. When this construct was transformed into A. niger, no detectable PLA2 was produced. Evidence was obtained showing that the PLA2 gene was transcribed and that PLA2 is extremely susceptible to both intracellular and extracellular proteases of A. niger, thus indicating that translation products would be rapidly degraded. By fusing the proPLA2-encoding sequence to the entire glaA gene, secreted yields of PLA2 up to 10 micrograms/ml were obtained from a transformed protease-deficient strain of A. niger. PLA2 was secreted in young cultures as a fusion protein, but in older cultures, it was processed from the glucoamylase carrier protein. Secreted PLA2 was shown to be enzymatically active and to have the correct N-terminal amino acid (aa) sequence, although another form of processed PLA2 was also produced. This form included two aa of the proregion from PLA2. The potential for improving yields of secreted heterologous proteins from A. niger still further is discussed.

Expression of a Brassica isopropylmalate synthase gene in Arabidopsis perturbs both glucosinolate and amino acid metabolism.

Isopropylmalate synthase (IPMS) is a key enzyme in the biosynthesis of the essential amino acid leucine, and thus primary metabolism. In Arabidopsis, the functionally similar enzyme, methythiolalkylmalate synthase (MAM), is an important enzyme in the elongation of methionine prior to glucosinolate (GSL) biosynthesis, as part of secondary metabolism. We describe the cloning of an IPMS gene from Brassica, BatIMS, and its functional characterisation by heterologous expression in E. coli and Arabidopsis. Over expression of BatIMS in Arabidopsis resulted in plants with an aberrant phenotype, reminiscent of mutants in GSL biosynthesis. Metabolite analyses showed that these plants had both perturbed amino acid metabolism and enhanced levels of GSLs. Microarray profiling showed that BatIMS over expression caused up regulation of the genes for methionine-derived GSL biosynthesis, and down regulation of genes involved in leucine catabolism, in addition to perturbed expression of genes involved in auxin and ethylene metabolism. The results illustrate the cross talk that can occur between primary and secondary metabolism within transgenic plants.

An ELISA method for the rapid and simple determination of aflatoxin in human serum

A rapid, simple method for the determination of aflatoxin in human serum using anti-aflatoxin antisera in an indirect, double antibody microtitration plate enzyme-linked immunosorbent assay (ELISA) is described. Direct assay of serum necessitated the availability of a large sample volume, due to the presence of non-specific interference. Addition of methanol to sera removed the interference enabling the analysis to be performed on small volumes (0.5 ml) of serum. The sensitivity of the assay is 20 pg ml-1. Total assay time, including sample preparation, is 4.5 h. No aflatoxin was found in any of the UK samples analysed.

Measurement of aflatoxin in Nigerian sera by enzyme-linked immunosorbent assay

Sera from 78 healthy men donating blood in Enugu, Nigeria were examined for aflatoxin by enzyme-linked immunosorbent assay (ELISA). Levels varied from less than 20 pg/ml to 3.1 ng/ml. The ELISA method is simple, sensitive and specific, and therefore well suited to the low-resource tropical environment. Aflatoxin is ingested in considerable amounts in Nigeria and many contribute, with hepatitis B, to the development of hepatocellular carcinoma.

Analysis of UK Sera for Aflatoxin by Enzyme-linked Immunosorbent Assay

1 Aflatoxins are toxic, carcinogenic secondary fungal metabolites produced by certain moulds that commonly infest foods. Measurement of aflatoxins in human serum would give a direct measurement of exposure. 2 Twenty-seven serum samples from UK blood donors were found to contain aflatoxin levels not greater than 64 pmol/1 (20 pg/ml) by an enzyme-linked immunosorbent assay. 3 These findings may indicate that present UK guideline tolerances for aflatoxin in imported food are effective in limiting human exposure to toxic aflatoxins in the UK diet, though further work would be needed to confirm this. In particular, sub-populations suspected of being at higher risk may need special considerations.

Immunoassay of Aflatoxin in Food and Human Tissue

AbstractAn enzyme-linked immunosorbent assay (ELISA) developed to determine aflatoxin in food was adapted to analyse rapidly human serum for aflatoxin.Sera from subjects in the U.K., Nigeria and Nepal were studied. No aflatoxin was found in U.K. sera, whilst 76% and 100% respectively of Nigerian and Nepalese samples were found positive for aflatoxin.Study was also made of maternal and cord sera from Thai subjects. Only 6% of maternal blood had detectable aflatoxin whilst 49% of cord sera were found positive for aflatoxin. This is evidence of trans-placental transfer of aflatoxin in humans and possibly of concentration of aflatoxin by the feto-placental unit.

High Serum Concentrations of Aflatoxin in Nepal as Measured by Enzyme-Linked Immunosorbent Serum Assay

1 Sera from 28 Nepalis, both patients and workers in a hospital in Banepa, Nepal were examined for AFB1 by enzyme-linked immunosorbent assay (ELISA). 2 This assay, previously validated using spiked sera, provides a sensitive rapid determination of serum aflatoxin (B1, G1 and Q1). 3 All 28 sera were positive with concentrations from 60 pg ml-1 to 10 ng ml-1 . 4 These results suggest that consumption of aflatoxin in Nepal is high (> 50-800 ng kg -1 d-1). 5 No reports of the degree of contamination of food, human consumption or body fluid concentrations of aflatoxin in Nepal have been previously published. 6 Aflatoxin may contribute to the development of hepatocellular carcinoma, which is probably a common tumour in Nepal.