The uses of computer-aided signal peptide selection and polymerase chain reaction in gene construction and expression of secreted proteins.
The computer program, SIGSEQ2, was used to select heterologous signal peptides from a catalog of published sequences to express the echistatin gene in insect (Sf9) cells. S-values for each amino acid were determined to select empirically the site of cleavage between the signal peptide and mature echistatin. Five gene fragments encoding the signal peptides for human immunoglobulin kappa (Ig kappa), Drosophila 68C glue, antistasin, bovine growth hormone (bGH), and human apolipoprotein E (Apo E) were constructed by the use of long synthetic oligonucleotides or polymerase chain reaction (PCR). Echistatin expression vectors then were constructed using the baculovirus polyhedrin promoter. Following transient transfection, the media were assayed for echistatin activity. The results indicate that the computer program greatly facilitated the selection and design of five different signal peptides and accurately predicted their relative functionality in the expression and secretion of echistatin in insect cell cultures.
Daugherty
B L
BL
Department of Cellular and Molecular Biology, Merck, Sharp & Dohme Research Laboratories, Rahway, NJ 07065.
Zavodny
S M
SM
Lenny
A B
AB
Jacobson
M A
MA
Ellis
R W
RW
Law
S W
SW
Mark
G E
GE
D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D005813:Genes, Synthetic; D006133:Growth Substances / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins; D007447:Invertebrate Hormones / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals; D012984:Software; D014162:Transfection; D014757:Viper Venoms / Q000235:genetics
D001057:Apolipoproteins E; D006133:Growth Substances; D007145:Immunoglobulin kappa-Chains; D036341:Intercellular Signaling Peptides and Proteins; D007447:Invertebrate Hormones; D010455:Peptides; D021382:Protein Sorting Signals; D014757:Viper Venoms; C053155:antistasin; C057986:echistatin