Chemical synthesis, cloning and expression of human epidermal growth factor gene in Saccharomyces cerevisiae.
A gene coding for human epidermal growth factor (hEGF) has been chemically synthesized by solid-phase phosphoramidite method. The 173 base-pair synthetic DNA duplex consists of a structural gene encoding hEGF, a stop codon TGA at 3' end and some convenient restriction sites at both ends of the gene. The synthesis of the gene involved enzymatic joining of 8 oligonucleotides to form a DNA duplex which was cloned into vector M13mp18. The recombinant colonies were identified by dot hybridization and restriction enzyme digestion. Its accuracy was confirmed by DNA sequence analysis. The hEGF DNA was inserted into yeast secretory vector YFD59. The resulting expression plasmid YFD104 was introduced into yeast Saccharomyces cerevisiae. The binding assay showed that the yeast transformants could express and secrete hEGF.
Yuan
H
H
Institute of Genetics, Fudan University, Shanghai, China.
Min
Y
Y
Shi
S
S
Tao
W
W
Wu
S
S
Li
Y
Y
Wang
Q
Q
Lu
H
H
Zhang
C
C
Hu
Q
Q
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004815:Epidermal Growth Factor / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D009841:Oligonucleotides; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D004262:DNA Restriction Enzymes