Production of biologically active recombinant tilapia insulin-like growth factor-II polypeptides in Escherichia coli cells and characterization of the genomic structure of the coding region.
Insulin-like growth factor-II (IGF-II) is a fetal growth factor in humans, but has not been clearly identified in fish up to now. For a detailed understanding of the physiological response of fish IGF-II, the first step was to clone tilapia IGF-II cDNA from the brain cDNA library, coding the region of genomic DNA, and also expressing tilapia IGF-II polypeptides from Escherichia coli. Tilapia cDNA sequences total 1,977 bp, and predicted nucleotide sequences and amino acid sequences of tilapia share 77.9% and 90.7% homology identity with rainbow trout IGF-II, respectively. The genomic structure of the tilapia prepro-IGF-II coding region is very difficult to sequence in mammals and birds. The cloned tilapia IGF-II gene coding region appears much more complex than in other vertebrates. In tilapia IGF-II, the first coding exon I encoding part of the signal peptide sequence is 25 amino acids shorter than the first coding exon of mammals and birds. The other 23 amino acids of the signal peptide, and the first amino acids of the B domain and C domain are encoded by tilapia coding exon 2. The C, A, and D domains, and the first 20 amino acids of the E peptide are encoded by tilapia coding exon 3. The other E peptides and the 3' untranslated region (UTR) region are encoded by tilapia coding exon 4. These data show that the IGF-II genes have significantly differing structures in vertebrate evolution, and there are differences of interrupting introns in the IGF-I genomic structure compared with mammals. To obtain recombinant biologically active polypeptides, tilapia IGF-II B-C-A-D domains were amplified using the polymerase chain reaction (PCR), then ligated with glutathione S-transferase (GST, pGEX-2T vector). Tilapia recombinant IGF-II protein was purified and characterized in E. coli. The fusion protein was also digested with thrombin and appeared as a recombinant IGF-II polypeptide single band with a molecular mass of 7 kD. The recombinant tilapia IGF-II protein biological function was measured by stimulation of [3H]thymidine incorporation. The assay concentration was set up from 0 to 120 nM to stimulate tilapia ovary cell line (TO-2) significantly to uptake thymidine. The results suggest that the recombinant IGF-II protein was dose dependent.
Chen
J Y
JY
Institute of Zoology, National Taiwan University, Taipei, R.O.C.
Chang
C Y
CY
Chen
J C
JC
Shen
S C
SC
Wu
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D002455:Cell Division; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005260:Female; D005796:Genes / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010053:Ovary; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D017210:Tilapia
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D007335:Insulin-Like Growth Factor II; D004247:DNA; D005982:Glutathione Transferase