Cloning and expression of a gene encoding a protein obtained from earthworm secretion that is a chemoattractant for garter snakes.
The protein ES20, derived from earthworm shock secretion, is a vomeronasally mediated chemoattractant for garter snakes (Jiang, X. C., Inouchi, J., Wang, D., and Halpern, M. (1990) J. Biol. Chem. 265, 8736-8744). Based on its 15-residue N-terminal amino acid sequence, degenerative oligodeoxynucleotide probes were synthesized and used to screen a cDNA library that was constructed in sense orientation using a Uni-ZAPTM XR vector and XL1-Blue MRF' host. A gene was cloned from a polymerase chain reaction as well as from the cDNA library. A combination of the forward degenerative primer and T7 primer was used to obtain gene-specific DNA fragments, from which probes were synthesized and successfully used in screening the cDNA library. The ES20 gene is about 700 base pairs long and encodes 208 amino residues. The ES20 gene was excised from a recombinant plasmid pSK-ES20, ligated to pQE30 expression vector, and transformed into Escherichia coli strain JM109. The selected recombinant plasmids were transformed into expression host cell, E. coli M15[pREP4]. Three transformants were selected, induced with isopropyl-1-thio-beta-D-galactopyranoside for fusion gene expression and an expressed 20-kDa fusion protein purified under denaturing conditions. This protein was refolded and gave a positive reaction against ES20-specific polyclonal antibodies. The fusion protein that had not been denatured remained as an aggregate and was an active chemoattractant for garter snakes.
Liu
W
W
Department of Biochemistry, SUNY Health Science Center at Brooklyn, New York 11203, USA.
Wang
D
D
Chen
P
P
Halpern
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002630:Chemotactic Factors / Q000096:biosynthesis* / Q000235:genetics*; D019976:Cloning, Organism; D017812:Colubridae; D018076:DNA, Complementary; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D002630:Chemotactic Factors; D018076:DNA, Complementary; C108946:ES20 protein, Lumbricus terrestris; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins