The light chain of HLA class I protein (β2m) has been expressed in Aspergillus nidulans. The cDNA of β2m was modified using the polymerase chain reaction to include overlapping extensions for its subsequent fusion into an Aspergillus vector. This fusion resulted in β2m cDNA being flanked by the Aspergillus awamori glucoamylase promoter and the Aspergillus niger glucoamylase terminator. Expression of β2m was induced by the addition of starch to the culture medium. In preliminary mass culture trials, 117 μg/liter of fβ2m were obtained in 60-liter fermentations. N-terminal sequencing of purified human β2m produced in fungi (fβ2m) revealed that 28% of the purified protein was of proper sequence and 61% of the protein had an additional serine anti lysine residue derived from the C-terminus of the fungal leader. Purified fβ2m from culture supernatants appeared biochemically similar to β2m obtained from human urine (uβ2m) as seen in immunoblot analysis. Functionally, fβ2m effectively interacted as a subunit of class I MHC molecules. This was seen both in a sandwich ELISA for detecting properly folded HLA class I heavy chain and in assays showing cell- surface β2m exchange into the mouse class I MHC H-2K(d). In these experiments the biological activity of fβ2m was indistinguishable from uβ2m. The successful expression of biologically active β2m in A. nidulans suggests that fungal systems might be useful for the production of other active components of the HLA class I MHC complex.
|Original language||English (US)|
|Number of pages||10|
|State||Published - Dec 1996|
ASJC Scopus subject areas
- Immunology and Allergy