Molecular cloning, sequencing and expression in Escherichia coli of the 25-kDa growth-related protein of Ehrlich ascites tumor and its homology to mammalian stress proteins.
The growth-related 25-kDa protein (p25) of Ehrlich ascites tumor (EAT) has been characterized by molecular cloning and sequencing of cDNA clones detected by hybridization with oligonucleotide probes synthesized according to the amino acid sequence of a tryptic peptide of p25. Detection of p25 mRNA in EAT of the exponential growth phase and of the stationary phase using cDNA-derived RNA probes demonstrated that the abundance of p25 mRNA is also growth-related. High-level expression of p25 in Escherichia coli has been established by oligonucleotide-directed mutagenesis of cDNA and insertion of the mutated cDNA into a T7-promoter expression vector. Recombinant p25 from the expressed cDNA sequence has been shown to comigrate with EAT p25 in electrophoresis and to react with antibodies against the EAT p25. On the amino acid level, p25 shows about 80% sequence homology to the human stress protein hsp27. Furthermore, p25 has similar isoforms of phosphorylation as demonstrated for small mammalian stress proteins from rat and human. From the results obtained, it is concluded that p25 is a mammalian stress protein, the abundance of which is related to growth characteristics of the Ehrlich ascites tumor.
Gaestel
M
M
Central Institute of Molecular Biology, Academy of Science of German Democratic Republic, Berlin-Buch.
Gross
B
B
Benndorf
R
R
Strauss
M
M
Schunk
W H
WH
Kraft
R
R
Otto
A
A
Böhm
H
H
Stahl
J
J
Drabsch
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002286:Carcinoma, Ehrlich Tumor / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000032:analysis*; D051379:Mice; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014357:Trypsin
D000596:Amino Acids; D006360:Heat-Shock Proteins; C486113:Hsbp1 protein, mouse; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D014357:Trypsin