Expression of a functional thyroglobulin fragment in a baculovirus system.
The synthesis is described of an N-terminal thyroglobulin (Tg) polypeptide of 27 kDa, which is capable of hormonogenesis, in a baculovirus system. This polypeptide was made using a 657 bp Tg cDNA cloned from human thyroid RNA by a polymerase chain reaction method. The cDNA contained the information for the Tg signal peptide, the N-terminally located site for thyroid hormone formation and, at the 3' end, a sequence coding for six histidine residues. The fragments produced were purified using a nickel-nitrilotriacetic acid column using these six histidine residues. The products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and showed two glycosylated fragments of 32 and 34 kDa, both of which started with asparagine. Iodination of the fragments with lactoperoxidase in vitro resulted in the formation of thyroxine (T4). The formation rate of T4 in the fragments was about five times lower than that of the mature Tg dimer of 660 kDa, but ten times more rapid than in bovine serum albumin under the same conditions.
den Hartog
M T
MT
Department of Endocrinology, University of Amsterdam, The Netherlands.
Sijmons
C C
CC
Kristel
P M
PM
Bakker
O
O
de Vijlder
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D007455:Iodine / Q000378:metabolism; D007784:Lactoperoxidase / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013954:Thyroglobulin / Q000096:biosynthesis* / Q000235:genetics; D013974:Thyroxine / Q000096:biosynthesis
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013954:Thyroglobulin; D007455:Iodine; D007784:Lactoperoxidase; D013974:Thyroxine