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

Abstract

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.Various genes and corresponding proteins are known to be expressed in a cell-cycle-dependent or, more generally, growthdependent manner, i.e. the levels of specific mRNAs and proteins vary over a cell cycle and in different growth phases of cell populations (for reviews see [l, 21).It is known that after transplantation of the Ehrlich ascites tumor (EAT) in mice the tumor shows exponential growth. Later on, the growth rate of the tumor declines continuously and EAT ultimately reaches a stationary phase without any further increase in tumor mass. Recently, it has been shown that the different stages of development of EAT in mice are correlated with changes in the synthesis as well as in the abundance of several proteins [3,4]. One of the most striking changes in the abundance of EAT proteins during tumor growth is the accumulation of a 25-kDa protein (p25) and its differently phosphorylated isoforms within the stationary phase of the EAT [3-51, in which cells are preferentially in the late S and G2 phase [6].In this paper we describe molecular cloning, sequencing and expression in Escherichiu coli of the growth-related 25-kDa EAT protein (p25). From the data obtained, the homology of p25 to small mammalian stress proteins is shown. MATERIALS AND METHODS Purification of p25The nonphosphorylated isoform of p25 was purified by preparative two-dimensional electrophoresis ('giant' gels) [7] with the modified conditions described by Neville [8] for the second dimension. Tryptic digestion and peptide sequence analysisEAT p25 (4 nmol) was digested with trypsin in 80 p10.1 M N-ethylmorpholine acetate pH 8.1 for 4 h at 37°C (enzyme/ substrate ratio: 1:50). The digest was acidified with 10% trifluoroacetic acid to pH 2 and separated by HPLC on a Vydac C-4 column (4.6 x 75 mm) using a linear gradient of 0 -60% acetonitrile in 0.05% t...

Overexpression of murine small heat shock protein HSP25 interferes with chondrocyte differentiation and decreases cell adhesion

“…It leads to a transgene transcript of about 1000 nt. Both bands display higher molecular size than the endogenous HSP25 mRNA, which is about 900 nt long 34,35 as seen in the F9 cell extract (lane 6) in which HSP25 is constitutively expressed. A third, smaller (less than 900 nt) and fainter, signal is detected in clone S4-2.…”

Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins

“…MAPKAP kinase-2 [1], recombinant marine hsp25 [21] and recombinant hsp27 [22] were purified as described. MAPKAP kinase-I (Mr. C. Satherland) [23] and the catalytic subunit of protein phosphatase 2A (Dr. D. Schelling) [24] were purified from rabbit skeletal muscle at Dundee by the investigators in parentheses.…”

Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen‐activated protein (MAP) kinase and MAP‐kinase‐activated kinases 1 and 2

“…CaM kinase-11, purified from rat brain, was provided by Dr A. Nairn (Rockefeller University, New York) and the mixed isoforms of the 14.3.3 protein (purified from sheep brain [30]) by Dr. A. Aitken (National Institute for Medical Research, London). Murine hsp2.5 [35] (provided by Dr M. Gaestel, Max Delbruck Centre for Molecular Medicine, Berlin, Germany) and a glutathione-transferase-MAPkinase fusion protein 1231 were also purified to homogeneity after expression in Escherichia coli. The MAP kinase was then activated using MAP kinase kinase.…”