Expression and characterization of recombinant human type II collagens with low and high contents of hydroxylysine and its glycosylated forms.
Insect cells coinfected with two baculoviruses, one coding for the pro alpha chains of human type II procollagen and the other for both the alpha and beta subunits of human prolyl 4-hydroxylase, produced the cartilage-specific type II collagen with a stable triple helix. The highest expression levels, up to 50 mg/l of type II collagen, were obtained in suspension culture using a modified construct in which sequences coding for the signal peptide and N propeptide of type II procollagen had been replaced by those for type III procollagen. The type III N propeptide artificially generated into type II procollagen was found to be cleaved at a much higher rate than the wild-type type II N propeptide, probably because the former interacted poorly with the triple-helical domain of type II procollagen. The amino acid composition of the recombinant type II collagen was very similar to that of the non-recombinant protein, but the hydroxylysine content was only 17% and that of glycosylated hydroxylysines was equally low. The hydroxylysine content was increased to the level found in the non-recombinant collagen by using an additional baculovirus coding for lysyl hydroxylase, and a substantial increase was also found in the glycosylated hydroxylysine content. No difference in thermal stability was found between the low- and high-hydroxylysine collagens.
Nokelainen
M
M
Collagen Research Unit, University of Oulu, Finland.
Helaakoski
T
T
Myllyharju
J
J
Notbohm
H
H
Pihlajaniemi
T
T
Fietzek
P P
PP
Kivirikko
K I
KI
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D003094:Collagen / Q000096:biosynthesis* / Q000737:chemistry; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006901:Hydroxylysine / Q000032:analysis*; D017924:Nucleopolyhedroviruses; D011347:Procollagen / Q000096:biosynthesis / Q000235:genetics; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000235:genetics / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry; D018411:Spodoptera; D013696:Temperature
D011347:Procollagen; D011994:Recombinant Proteins; D006901:Hydroxylysine; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; D005964:Glucosyltransferases; C023247:UDP glucose-collagen glucosyltransferase