Recombinant mouse prolactin: expression in Escherichia coli, purification and biological activity.
Transformation of Escherichia coli cells with a recombinant plasmid containing modified mouse prolactin (mPRL) cDNA and a pKK223-3 vector resulted in efficient expression of mPRL protein. Cloned mPRL cDNA was modified by removing the 5' non-translating sequence as well as the sequence which encoded the signal peptide of preprolactin for recombination. In addition, approximately 100 nucleotides of the 5'-terminal region of the cDNA, which include the ATG initiation codon and the following 31 codons of mature mPRL, were replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen to be rich in AT without changing the amino acid sequence of the protein. The modified cDNA was finally inserted into the multicopy plasmid, pUC19, before high-level expression of mPRL in E. coli cells was obtained. Western blotting analysis of total protein from transformed E. coli cells showed that both 23 and 16 kDa peptides were recognized by specific mPRL antisera. The purified and refolded 23 kDa protein exhibited a growth-stimulating effect on rat Nb 2 Node lymphoma cells, and was very similar to that of natural pituitary PRL.
Yamamoto
M
M
Central Research Laboratories, Shikibo Ltd, Osaka, Japan.
Harigaya
T
T
Ichikawa
T
T
Hoshino
K
K
Nakashima
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010957:Plasmids; D011388:Prolactin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D009841:Oligonucleotides; D011994:Recombinant Proteins; D011388:Prolactin