Gene sequence, cDNA construction, expression in Escherichia coli and genetically approached purification of porcine interleukin-1 beta.
A genomic clone (PIL3) covering the 8.8-kb prointerleukin-1 beta ('catabolin') gene of the domesticated swine (Sus scrofa domestica) was isolated from a genomic library and characterized by nucleotide sequencing. Typical features of the gene include a seven-exon structure, with the highest degree of nucleotide and amino acid conservation among human and porcine genes being found in the receptor-binding portion encoded by exons six and seven. Three 250-bp repetitive elements with a > 75% similarity to the pig repetitive element-1 family sequence are located in untranslated gene segments. Southern-hybridization experiments disclosed extensive genomic heterogeneity of the porcine interleukin-1 beta (IL-1 beta) gene region, suggesting a duplication of at least the 3' half of the gene in the porcine genome. Since similar hybridization patterns were observed for wild boar (Sus scrofa) genomic DNA, it was concluded that this gene rearrangement had preceded domestication of the wild swine. In addition, the cDNA for processed porcine IL-1 beta was constructed through polymerase-chain-reaction-mediated exon fusion by overlap extension starting from the genomic template. Recombinant IL-1 beta was expressed in Escherichia coli as a fusion protein containing an N-terminal hexahistidine tag followed by a factor-Xa-cleavage site. The protein was efficiently purified through adoption of a scheme that consisted of four alternating cycles of immobilized metal-ion-affinity chromatography and size-exclusion chromatography. 13.8 mg highly purified recombinant porcine IL-1 beta was obtained starting from a 900-ml thermo-induced E. coli culture (final endotoxin concentration < 0.22 ng/ml). The protein behaved homogeneously as a monomeric species, which was reactive in Western-blot experiments with an anti-(human-IL-1 beta) serum and which appeared to induce gelatinase B in MDBK cells in a dose-dependent fashion.
Vandenbroeck
K
K
Rega Institute, University of Leuven, Belgium.
Fiten
P
P
Beuken
E
E
Martens
E
E
Janssen
A
A
Van Damme
J
J
Opdenakker
G
G
Billiau
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D007375:Interleukin-1 / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D012045:Regulatory Sequences, Nucleic Acid; D013552:Swine / Q000235:genetics*
D018076:DNA, Complementary; D007375:Interleukin-1; D011993:Recombinant Fusion Proteins; D004247:DNA