Abstract
A DNA sequence coding for human muscle acylphosphatase has been constructed using 16 chemically synthesized oligonucleotides. The 300-bases long DNA sequence has been cloned in the pT7.7 Escherichia coli expression vector and in the pYEpsec1 Saccharomyces cerevisiae expression vector. In both cases a high level of expression of acylphosphatase has been observed. The recombinant proteins have been purified to homogeneity and assayed in comparison with the natural protein, using benzoylphosphate as a substrate and phosphate as a competitive inhibitor. The recombinant enzymes expressed in the two microorganisms maintain the kinetic properties of the natural protein. In addition, NMR analysis shows that the gross fold of the two recombinant enzymes is correct.
| Original language | English |
|---|---|
| Pages (from-to) | 369-374 |
| Number of pages | 6 |
| Journal | BBA - Gene Structure and Expression |
| Volume | 1216 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Dec 14 1993 |
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Keywords
- (Human)
- Gene expression
- NMR
- Recombinant acylphosphatase
- Synthetic gene
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Genetics
- Structural Biology
Cite this
Chemical synthesis and expression of a gene coding for human muscle acylphosphatase. / Modesti, Alessandra; Raugei, Giovanni; Taddei, Niccolo'; Marzocchini, Riccardo; Vecchi, Manuela; Camici, Guido; Manao, Giampaolo; Ramponi, Giampietro.
In: BBA - Gene Structure and Expression, Vol. 1216, No. 3, 14.12.1993, p. 369-374.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Chemical synthesis and expression of a gene coding for human muscle acylphosphatase
AU - Modesti, Alessandra
AU - Raugei, Giovanni
AU - Taddei, Niccolo'
AU - Marzocchini, Riccardo
AU - Vecchi, Manuela
AU - Camici, Guido
AU - Manao, Giampaolo
AU - Ramponi, Giampietro
PY - 1993/12/14
Y1 - 1993/12/14
N2 - A DNA sequence coding for human muscle acylphosphatase has been constructed using 16 chemically synthesized oligonucleotides. The 300-bases long DNA sequence has been cloned in the pT7.7 Escherichia coli expression vector and in the pYEpsec1 Saccharomyces cerevisiae expression vector. In both cases a high level of expression of acylphosphatase has been observed. The recombinant proteins have been purified to homogeneity and assayed in comparison with the natural protein, using benzoylphosphate as a substrate and phosphate as a competitive inhibitor. The recombinant enzymes expressed in the two microorganisms maintain the kinetic properties of the natural protein. In addition, NMR analysis shows that the gross fold of the two recombinant enzymes is correct.
AB - A DNA sequence coding for human muscle acylphosphatase has been constructed using 16 chemically synthesized oligonucleotides. The 300-bases long DNA sequence has been cloned in the pT7.7 Escherichia coli expression vector and in the pYEpsec1 Saccharomyces cerevisiae expression vector. In both cases a high level of expression of acylphosphatase has been observed. The recombinant proteins have been purified to homogeneity and assayed in comparison with the natural protein, using benzoylphosphate as a substrate and phosphate as a competitive inhibitor. The recombinant enzymes expressed in the two microorganisms maintain the kinetic properties of the natural protein. In addition, NMR analysis shows that the gross fold of the two recombinant enzymes is correct.
KW - (Human)
KW - Gene expression
KW - NMR
KW - Recombinant acylphosphatase
KW - Synthetic gene
UR - http://www.scopus.com/inward/record.url?scp=0027724574&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027724574&partnerID=8YFLogxK
U2 - 10.1016/0167-4781(93)90003-V
DO - 10.1016/0167-4781(93)90003-V
M3 - Article
C2 - 8268218
AN - SCOPUS:0027724574
VL - 1216
SP - 369
EP - 374
JO - Biochimica et Biophysica Acta - Gene Structure and Expression
JF - Biochimica et Biophysica Acta - Gene Structure and Expression
SN - 0167-4781
IS - 3
ER -