Construction And Expression Of Red Fluorescent Protein Fusion Vector Of P38 MAPK

AbstractMitogen-activated protein kinases (MAPKs) composing one of the important signal transduction systems in organisms are involved in many cellular processes, such as cell growth, development, division, differentiation, death and coordination of cellular functions, etc. Four subfamilies of MAP kinases, i.e., ERK, JNKISAPK, p38/RK and ERK5/BMK1, have been identified and cloned in mammalian cells. p38 MAPK is the central component of the p38 signal transduction pathway. So far, four p38 isoforms, which are p38a, p38f~, p38y and p38? have been cloned and characterized. It had been shown that the activation of p38 had effects on the cellular processes of cell growth, cell cycle and apoptosis, and also involved in the regulation of inflammation and stress responses stimulated by lipopolysaccharide (LPS). The study on the cellular localization of protein kinases is helpful for the elucidation of enzyme specific function. However, the intracellular localization of p38 MAPK is not well studied through direct method up to now. Fluorescent proteins, a family of proteins with bioluminescence function, may be used to directly observe intracellular localization of proteins. More recently, red fluorescent protein (RFP), a new member of fluorescent protein family, was found and then applied with advantages in scientific research at once. The ratio of signal-to-noise of RFP is higher than that of green fluorescent protein (GFP), which makes it a useful tool for the research of intracellular localization of proteins. To study the intracellular localization of p38 MAPK, RFP fusion vectors of four p38 isoforms was constructed by gene engineering, and expressed in mammalian cells. First, four FLAG-tagged p38 isoforms in pcDNA3 vector were amplified by polymerase chain reaction (PCR). Then the product of PCR was digested with restrictive endonucleases Hind III and BamH IIBgl II respectively, followed by ligation with pDsRedl-N1 vector which had been digested with Hind III and BamH I. The E. coli strain DH5c~ was transformed with the mixture of ligation. Then transformatiom colonies were picked at-5-random, and the recombinant plasmids were extracted. Having been verified via restrictive endonucleases digestion, PCR and DNA sequencing, the recombinant plasmids were transfected into HeLa and WEHI cells in 24-well plate, so as to investigate the application prospect of RFP fusion protein in research of intracellular localization of proteins. 36h after transfection, cells were fixed and observed with fluorescent microscope (excitation wavelength at 558nm, absorption wavelength at 583nm). The expression of four p38 isoforms in HeLa and WEHI cells was viewed and recorded by taking pictures. The results of transfection showed that four p38 isoforms all distributed all over the cytosol and nuclei in resting cells, suggesting that the RFP fusion expression vectors we constructed possess functions, and using the method of RFP fusion protein to study the intracellular localization of protein kinases is feasible as well. All in a word, the p38 RFP fusion vectors constructed by us provide a useful tool for elucidation of the intracellular localization and the mechanisms of translocation of p38 MAPK.