Expression of human recombinant beta 2-microglobulin by Aspergillus nidulans and its activity.
The light chain of HLA class I protein (beta 2m) has been expressed in Aspergillus nidulans. The cDNA of beta 2m was modified using the polymerase chain reaction to include overlapping extensions for its subsequent fusion into an Aspergillus vector. This fusion resulted in beta 2m cDNA being flanked by the Aspergillus awamori glucoamylase promoter and the Aspergillus niger glucoamylase terminator. Expression of beta 2m was induced by the addition of starch to the culture medium. In preliminary mass culture trials, 177 micrograms/liter of f beta 2m were obtained in 60-liter fermentations. N-terminal sequencing of purified human beta 2m produced in fungi (f beta 2m) revealed that 28% of the purified protein was of proper sequence and 61% of the protein had an additional serine and lysine residue derived from the C-terminus of the fungal leader. Purified f beta 2m from culture supernatants appeared biochemically similar to beta 2m obtained from human urine (u beta 2m) as seen in immunoblot analysis. Functionally, f beta 2m 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 beta 2m exchange into the mouse class I MHC H-2Kd. In these experiments the biological activity of f beta 2m was indistinguishable from u beta 2m. The successful expression of biologically active beta 2m in A. nidulans suggests that fungal systems might be useful for the production of other active components of the HLA class I MHC complex.
O'Herrin
S M
SM
Department of Surgery, University of Wisconsin-Madison 53792, USA.
Kulkarni
S
S
Kenealy
W R
WR
Fechner
J H
JH
Sollinger
H
H
Schneck
J P
JP
Burlingham
W J
WJ
D001233:Aspergillus nidulans / Q000235:genetics*; D001692:Biological Transport; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000494:pharmacology*; D001613:beta 2-Microglobulin / Q000032:analysis / Q000096:biosynthesis* / Q000494:pharmacology*
D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin