Production of recombinant human serum albumin from Saccharomyces cerevisiae.
Human serum albumin has been constitutively expressed in a Saccharomyces cerevisiae brewing yeast. After cell growth and disruption the product was associated with the insoluble fraction and represented approximately 1% of total cell protein. After the cell debris was extensively washed, the albumin was solubilized with 8 M urea and 28 mM 2-mercaptoethanol in 50 mM sodium carbonate buffer, pH 10. The denatured albumin was refolded by dialysis and further purified by anion exchange and gel filtration chromatography. Losses of renatured material could be reduced, or higher protein concentrations used during refolding, if the denatured product was purified by cation-exchange chromatography in urea prior to refolding. Apart from an additional N-terminal N-acetyl methionine, the refolded product proved identical to human serum albumin derived from plasma when compared by a variety of physical, chemical, and biological analytical methods.
Quirk
A V
AV
Delta Biotechnology Limited, Nottingham, United Kingdom.
Geisow
M J
MJ
Woodrow
J R
JR
Burton
S J
SJ
Wood
P C
PC
Sutton
A D
AD
Johnson
R A
RA
Dodsworth
N
N
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide; D010450:Endopeptidases; D005822:Genetic Vectors; D006801:Humans; D008666:Metalloendopeptidases; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012709:Serum Albumin / Q000096:biosynthesis* / Q000302:isolation & purification; D013053:Spectrophotometry; D014357:Trypsin
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012709:Serum Albumin; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D003488:Cyanogen Bromide