Cloning, expression and purification of recombinant cotton rat interleukin-5.
The coding sequence of the hispid cotton rat (Sigmodon hispidus) interleukin-5 (IL-5) was isolated by a combination of reverse transcription (RT)-PCR and RACE protocols from concanavalin A stimulated spleen cells. The open reading frame of 399 bp encodes a polypeptide of 132 amino acids. Comparison with the rat, mouse, gerbil and human counterparts revealed 88, 88, 87 and 75% identity at the nucleotide level and 88, 90, 89 and 70% at the amino acid level, respectively. The entire coding sequence, minus the putative signal peptide sequence, was inserted into an inducible Escherichia coli expression vector. The recombinant protein possessed an expected molecular mass of 14kDa and was located in bacterial inclusion bodies. A purification scheme under reducing and denaturing conditions followed by subsequent successive dialysis steps led to the recovery of a recombinant dimeric cotton rat IL-5. The biological activity of the recombinant protein was demonstrated in a murine cell line proliferation assay. This activity was specifically inhibited by rat monoclonal antibodies directed against mouse IL-5. Together with specific antibodies that can be generated easily, cotton rat IL-5 constitutes a useful tool for extending the use of the cotton rat animal model in the study of various human pathogens.
Houard
S
S
Service de Génétique Appliquée, Université Libre de Bruxelles, avenue des Professeurs Jeener et Brachet 12, B-6041, Gosselies, Belgium. shouard@sga.ulb.ac.be
Jacquet
A
A
Haumont
M
M
Daminet
V
V
Milican
F
F
Glineur
F
F
Bollen
A
A
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D015848:Interleukin-5 / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016205:Sigmodontinae / Q000235:genetics*
D018076:DNA, Complementary; D015848:Interleukin-5; D011993:Recombinant Fusion Proteins