Secretory expression of human albumin domains in Saccharomyces cerevisiae and their binding of myristic acid and an acylated insulin analogue.
Albumin is organized in three homologous domains formed by double loops stabilized by disulfide bonds. Utilizing a secretory expression system based on a synthetic secretory prepro-leader, the three human serum albumin domains were expressed in the yeast Saccharomyces cerevisiae. Human serum albumin domains I and III were efficiently expressed and secreted, indicating that these domains can form independent structural units capable of folding into stable tertiary structures. In contrast, albumin domain II was not secreted and disappeared early in the secretory pathway. Human serum albumin has the ability to bind a large number of small molecule ligands, including fatty acids, presumably due to its structure and structural flexibility. Purified albumin domain III bound myristic acid, whereas purified albumin domain I did not bind myristic acid. A new soluble long-acting insulin an alogue acylated with myristic acid (Markussen J., et al., Diabetologia 39, 281-288, 1996) bound to domain III and bound markedly more weakly to domain I.
Novo Nordisk, Bagsvaerd, DK-2880, Denmark. thk@novo.dk
T
T
Kjeldsen
A F
AF
Pettersson
L
L
Drube
P
P
Kurtzhals
I
I
Jonassen
S
S
Havelund
P H
PH
Hansen
J
J
Markussen
D002352:Carrier Proteins / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism; D000069057:Insulin Detemir; D049528:Insulin, Long-Acting; D019814:Myristic Acid / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012709:Serum Albumin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D002352:Carrier Proteins; D007328:Insulin; D049528:Insulin, Long-Acting; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012709:Serum Albumin; D019814:Myristic Acid; D000069057:Insulin Detemir