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Production of human secretory component with dimeric IgA binding capacity using viral expression systems
L Rindisbacher1, S Cottet, R Wittek
1Institut de Biologie Animale, Bâtiment de Biologie, Université de Lausanne, Switzerland.
The Journal of Biological Chemistry
|June 9, 1995
Summary
Researchers developed recombinant methods to produce human secretory component (hSC), a key protein for immunoglobulin A. Both insect and mammalian cell systems efficiently secreted hSC, enabling its purification and functional analysis.
Area of Science:
- Molecular Biology
- Biochemistry
- Immunology
Background:
- The human polymeric immunoglobulin receptor (pIgR) plays a crucial role in the transport of dimeric IgA across epithelial cells.
- Secretory component (SC) is the extracellular portion of pIgR, essential for IgA function and stability.
- Efficient production of functional secretory component is vital for research and potential therapeutic applications.
Purpose of the Study:
- To develop and compare recombinant expression systems for producing soluble human secretory component (hSC).
- To assess the yield, purification, and functional integrity of recombinant hSC produced in insect and mammalian cells.
Main Methods:
- Insertion of cDNA encoding the extracellular domain of human pIgR into baculovirus and vaccinia virus transfer vectors.
- Expression of recombinant hSC in Sf9 insect cells and mammalian cell lines (CV-1, TK-143B, HeLa).
- Purification using concanavalin A or Ni(2+)-chelate affinity chromatography.
- Analysis of molecular weight and glycosylation patterns using endoglycosidase digestion.
- Assessment of functional reassociation with dimeric IgA.
Main Results:
- High-efficiency secretion of recombinant hSC into serum-free culture medium was achieved in both systems.
- The baculovirus/Sf9 insect cell system yielded up to 50 mg/L, while the vaccinia virus/mammalian cell system produced up to 10 mg/L.
- Recombinant hSC exhibited cell-line-dependent variations in molecular weight due to differential glycosylation.
- Single-step purification was feasible, and recombinant hSC retained its ability to bind dimeric IgA.
Conclusions:
- Recombinant baculovirus and vaccinia virus systems are effective for high-yield production of soluble human secretory component.
- The produced recombinant hSC is glycosylated differently depending on the expression system but remains functionally active.
- These systems provide a valuable source of recombinant hSC for further research and potential therapeutic development.