The expression of functional link protein in a baculovirus system: analysis of mutants lacking the A, B and B' domains.
Functional recombinant human link protein has been produced using a baculovirus expression system. In addition to the intact link protein, three mutant forms have also been expressed. Each mutant bears a deletion equivalent to the protein encoded by one exon in the gene. These deletions represent the A domain, which is thought to be responsible for interaction with aggrecan, and the B or B' domains, which are associated with the interaction with hyaluronate. Such deletions split codons spanning exon boundaries, but maintain the reading frame of the protein and result in the correct amino acid being present at the splice junction. All the recombinant proteins appear as two components upon SDS/PAGe, though the abundance of the two forms does vary between preparations, as a result of variable substitution by N-linked oligosaccharides. The recombinant intact link protein was able to interact with both hyaluronate and aggrecan, showing that the baculovirus system is able to produce functional molecules. All of the recombinant mutant link proteins were also able to interact with hyaluronate, indicating that both the B and B' domains can function independently. The recombinant mutant link proteins were also able to interact with aggrecan, with the exception of the mutant lacking the A domain, confirming that this ability resides entirely within this domain.
Grover
J
J
Genetics Unit, Shriners Hospital for Crippled Children, Montreal, Canada.
Roughley
P J
PJ
D053674:Aggrecans; D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D006801:Humans; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D009036:Moths; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010957:Plasmids; D011506:Proteins / Q000235:genetics* / Q000502:physiology; D011509:Proteoglycans / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D017384:Sequence Deletion; D000099249:Proteoglycan Link Protein
D053674:Aggrecans; D000581:Amidohydrolases; D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011506:Proteins; D011509:Proteoglycans; D011994:Recombinant Proteins; D000099249:Proteoglycan Link Protein