Affinity Bioadhesives Engineered for Cell Culture Applications

摘要

The objective of the program is to develop, through genetic engineeringtechnology, affinity bioadhesives to be used in applications requiring specific eucaryotic cell attachment. A series of recombinant adhesive proteins, based on the polyphenolic protein of the blue mussel, has been produced. The affinity bioadhesives are expected to retain the characteristics of the mussel adhesive protein and add specificity to the cell binding interactions. Hybrid proteins consisting of portions of the adhesive protein and the cell attachment region of fibronectin were prepared as the first affinity bioadhesives. Researchers have assembled the genetic constructions and engineered yeast cells to produce five different affinity bioadhesive proteins. Three of these proteins, chosen for their efficient expression in yeast, were produced in 10-liter fermentations and purified. The three hybrid proteins consist of variations in length and number of fibronectin cell attachment regions at the C-terminus of a 24,000 molecular weight portion of the mussel adhesive protein. One copy of the 30 amino acid fibronectin cell attachment region is present in AAP486 and two tandem copies are present in AAP492. AAP494 carries one copy of a 10 amino acid fibronectin cell attachment region. The three affinity bioadhesives were shown to have the same surface adhesion properties as the mussel adhesive proteins. Cell culture work with AAP486 suggests that some of the binding is through the fibronectin cell attachment region.