Cloning, expression and purification of recombinant cotton rat interferon-gamma.
The hispid cotton rat (Sigmodon hispidus) has proven to be an excellent small animal model; however, immunological studies have been limited due to a lack of available reagents. We report cloning of the cotton rat interferon-gamma (IFN-gamma) cDNA from concanavalin A-stimulated spleen cells using a combination of reverse transcription polymerase amplification reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) protocols. The open reading frame of 513 nucleotides encodes a 170 amino acid (aa) protein followed by a stop codon with a predicted molecular mass of 19548Da. Cotton rat IFN-gamma shares 63, 60, 43 and 43% identity with the hamster, gerbil, mouse and rat counterpart, respectively. IFN-gamma nucleotide sequence corresponding to aa 18-153 was expressed in Escherichia coli under tryptophan promoter control, either fused to a single initiating codon or fused to the thioredoxin coding sequence. Both expression products were found exclusively in bacterial inclusion bodies. Two purification schemes have been developed to purify the product fused to a single methionine. One of them is fast and leads to the recovery of a pure product suitable for use in antibody production. The second protocol, which includes chromatographic steps, allows the use of the purified product for in vitro demonstration of biological activity in a viral cytopathic reduction assay on cotton rat cells.
Houard
S
S
Service de Génétique Appliquée, Université Libre de Bruxelles, rue des Professeurs Jeener et Brachet 12, B-6041, Gosselies, Belgium. shouard@sga.ulb.ac.be
Jacquet
A
A
Haumont
M
M
Glineur
F
F
Daminet
V
V
Milican
F
F
Bollen
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007371:Interferon-gamma / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016205:Sigmodontinae / Q000235:genetics*; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects / Q000254:growth & development
D000998:Antiviral Agents; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007371:Interferon-gamma