Enhanced recovery and purification of Aspergillus glucoamylase from Saccharomyces cerevisiae by the addition of poly(aspartic acid) tails.
Poly(aspartic acid) tails of different lengths were fused to the glucoamylase (GA) of Aspergillus awamori by genetic engineering techniques. Tails consisting of 5, 7, and 10 aspartate residues were fused to the N-terminus of the full-length mature GA (aa 1-616) downstream from the intact leader peptide to produce fusion proteins designated GAND5, GAND7, and GAND10, respectively. Three fusion proteins with C-terminal tails were also constructed, designated GACD0, GACD5, and GACD10 (0, 5, and 10 aspartate residues, respectively). For the C-terminal fusion proteins, the tails were fused to a catalytically active but truncated form of GA (aa 1-484). All of the charged tails had the general sequence Met-Ala-Aspn-Tyr, where n = 0, 5, 7, or 10. The modified genes were expressed in the yeast Saccharomyces cerevisiae and the proteins secreted into the culture medium. The enzymes were subsequently purified by affinity chromatography. The specific activity of each purified enzyme was found to be comparable to the wild-type enzyme. The C-terminal tails did not interfere with expression, whereas decreased extracellular glucoamylase activities corresponding to increased tail length were found for the N-terminal fusion proteins. Amino-terminal amino acid sequence analysis of the purified GAND proteins confirmed the authenticity of the amino termini of the modified proteins and showed that both the leader peptidase and KEX2 protease cleavages had occurred faithfully. The increased net negative charge of the GAND and GACD proteins was indicated by both nondenaturing PAGE and isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)
Suominen
I
I
Department of Biochemistry, University of Turku, Finland.
Ford
C
C
Stachon
D
D
Heimo
H
H
Niederauer
M
M
Nurmela
H
H
Glatz
C
C
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics
D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C017645:polyaspartate; D005087:Glucan 1,4-alpha-Glucosidase