Molecular cloning of proteinase-encoding genes from cancer cells by zymogen assay and direct sequencing.
A method that permits the in vitro cloning and identification of proteolytic enzyme genes from cDNA expression libraries is described. The method can detect positive proteinase genes within 30 min following the transfer of plaques to nitrocellulose membrane filters. The method is based on the functional expression of fusion lac Z-proteinase protein in lambda gt11 infected Y1090 bacteria and does not require prior knowledge of either the sequence of the cDNA insert or a monoclonal antibody to its encoded antigen. This strategy when coupled with polymerase chain reaction of the cDNA insert using lac Z primer sequences that are flanking the EcoR1 cloning site in gt11 phage permits direct sequencing of the amplified DNA. With this method we have isolated 10 genes expressing protease activity in the human small-cell carcinoma of the lung. The same procedure could be applied to isolate unknown proteinases from cDNA libraries of virtually any eukaryotic cell.
Hagag
N G
NG
Division of Oncology, Health Sciences Center, State University of New York, Stony Brook 11794.
Kirchner
M
M
DiStefano
J F
JF
D001483:Base Sequence; D018288:Carcinoma, Small Cell / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004586:Electrophoresis; D010450:Endopeptidases / Q000235:genetics*; D004792:Enzyme Precursors; D015870:Gene Expression; D015723:Gene Library; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors; D010450:Endopeptidases