TY - JOUR
T1 - Synthesis of a gene for human serum albumin and its expression in Saccharomyces cerevisiae
AU - Kálmán, Miklós
AU - Cserpän, Imre
AU - Bajszár, György
AU - Dobi, Albert
AU - Horváth, Éva
AU - Pázmán, Cecilia
AU - Simoncsits, András
N1 - Funding Information:
Thanks are due to B. Aberg (Skandigen AB, Stockholm) and T. Bartfai (Stockholm University) for their continuous interest and useful discussions, and to H. Jornvall (Karolinska Institute.Stockholm) for the N-terminal amino acid sequencing performed in his laboratory. This work was largely supported by grants from Skandigen AB.
PY - 1990/10/25
Y1 - 1990/10/25
N2 - A 1761 base pairs long artificial gene coding for human serum albumin (HSA) has been prepared by a newly developed synthetic approach, resulting in the largest synthetic gene so far described. Oligonucleotides corresponding to only one strand of the HSA gene were prepared by chemical synthesis, while the complementary strand was obtained by a combination of enzymatic and cloning steps. 24 synthetic, 69-85 nucleotldes long oligonucleotides covering the major part of the HSA gene (41-1761 nucleotides) were used as building blocks. Generally, four groups of 6-6 such oligonucleotides were successively cloned in pUC19 Escherichia coli vector to obtain about quarters of the gene aslarge fragments. Joining of these four fragments resulted in a cloned DNA coding for the 13-585 amino acid region of HSA, which was further supplemented with a double-stranded linker sequence coding for the amino terminal 12 amino acids. The completed structural gene composed of frequently used codons in the highly expressed yeast genes was then supplied with yeast regulatory sequences and the HSA expression cassette so obtained was inserted into an Escherichla coll - Saccharomyces cerevisiae shuttle vector. This vector was shown to direct the expression in Saccharomyces cerevisiae of correctly processed, mature HSA which was recognized by antiserum to HSA, and possessed the correct N-terminalamino acid sequence.
AB - A 1761 base pairs long artificial gene coding for human serum albumin (HSA) has been prepared by a newly developed synthetic approach, resulting in the largest synthetic gene so far described. Oligonucleotides corresponding to only one strand of the HSA gene were prepared by chemical synthesis, while the complementary strand was obtained by a combination of enzymatic and cloning steps. 24 synthetic, 69-85 nucleotldes long oligonucleotides covering the major part of the HSA gene (41-1761 nucleotides) were used as building blocks. Generally, four groups of 6-6 such oligonucleotides were successively cloned in pUC19 Escherichia coli vector to obtain about quarters of the gene aslarge fragments. Joining of these four fragments resulted in a cloned DNA coding for the 13-585 amino acid region of HSA, which was further supplemented with a double-stranded linker sequence coding for the amino terminal 12 amino acids. The completed structural gene composed of frequently used codons in the highly expressed yeast genes was then supplied with yeast regulatory sequences and the HSA expression cassette so obtained was inserted into an Escherichla coll - Saccharomyces cerevisiae shuttle vector. This vector was shown to direct the expression in Saccharomyces cerevisiae of correctly processed, mature HSA which was recognized by antiserum to HSA, and possessed the correct N-terminalamino acid sequence.
UR - http://www.scopus.com/inward/record.url?scp=0025064548&partnerID=8YFLogxK
U2 - 10.1093/nar/18.20.6075
DO - 10.1093/nar/18.20.6075
M3 - Article
C2 - 2235491
AN - SCOPUS:0025064548
SN - 0305-1048
VL - 18
SP - 6075
EP - 6081
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 20
ER -