The Fusion Expression Of A Pea Actin Isoform (peac3) And Green Fluorescent Protein (gfp) And The Biochemical Characteristics Analysis To The Fusion Protein

Actins exist in eukaryotes generally. Under the control of various kinds of actin-binding proteins, actins participate in various cellular activities, such as cell division, endocytosis, nucleation, cell signaling, gravity sensing, and the tip growth, organelle movement etc. There are multiple actin isoforms which are encoded by the multi-gene family in plant cells; the actin isoforms expressed in tissue and organ specificity; and the actin isoforms play different functions in plant growth and development. In order to understand the function and dynamic regulation of the specific actin isoforms deeply, this study expressed and purified pea actin isoforms 3 (PEAc3) in prokaryotic expression system by GFP fusion technique and researched some biochemical characteristics of the fusionprotein, the main results are show as follows:Pea actin isoforms PEAc3 gene was cloned by RT-PCR method and compared with the seven kinds of other plant actins selected randomly, the homology was above 90%. Using DNAman (Ver6.0) bioinformatics software, a biological evolutionary tree was established among the pea actin isoforms which provided the support for the study of molecular evolution within eukaryotic. Based on the predicted amino acid sequence of PEAc3, the molecular weight (MW) of PEAc3 was 41.67kD; the pI was 5.17; PEAc3 was soluble protein without signal peptide sequence and exist as a mature protein, PEAc3 have three groups of sequence fingerprint which is the typical feature of actin; The predicted 3D map of PEAc3 is similar with that of skeletal muscle actin.In order to study the effect of gene fusion on PEAc3 prokaryotic expression, four prokaryotic expression vectors were constructed, that is PEAc3-pET30a, His-PEAc3-pET30a, His-PEAc3-GFP-pET30a and His-GFP-pET30a. The proteins expressed by above four vectors were analyzed by DNAman software:The length of PEAc3 was 377 amino acids, MW was 41.67kD, pI was 5.17; the length of His-PEAc3 was 427 amino acids, MW was 47.13kD, pI was 5.56; the length of His-GFP-PEAc3 was 675 amino acids, MW was 74.74kD, pI was 5.81; the length of His-GFP was 296 amino acids, MW was 33.04kD, pI was 6.26. The constructed vector was transformed into E. coli. BL21 by electroporation, the conditions of induced expression was optimized:PEAc3:the best bacterial concentration of adding IPTG was OD6oo=0.7, and the optimal IPTG concentration was 0.05mmol/L, induced time was 4h, temperature was 25℃, expressed in inclusion bodies mainly; His-PEAc3:the bacterial concentration of adding IPTG was OD6oo=0.5, and the optimal IPTG concentration was 0.1mmol/L, induced time was 3h, temperature was 37℃, expressed in inclusion bodies mainly; His-PEAc3-GFP:the best bacterial concentration of adding IPTG was OD6oo=0.8, and the optimal IPTG concentration was 0.1 mmol/L, induced time was 4h, temperature was 25℃, expressed in supernatant and inclusion bodies; His-GFP:the bacterial concentration of adding IPTG was OD6oo=0.5, and the optimal IPTG concentration was 0.05mmol/L, induced time was 4h, temperature was 37℃, expressed in supernatant mainly. The results showed that the fusion of GFP accelerated the soluble expression of PEAc3 in prokaryotic expression system.The four proteins above were purified by the denaturation and renaturation of urea, nickel column affinity chromatography and ion exchange chromatography etc. and some biochemical characteristics of the proteins were studied. By the comparison of polymerization curve and the critical polymerization concentration of PEAc3, His-PEAc3 and His-PEAc3-GFP, the polymerization kinetics of His-PEAc3-GFP is closer to that of the skeletal muscle actin. Determination of DNaseⅠactivity showed that, PEAc3, His-PEAc3 and His-PEAc3-GFP could inhibit the activity of DNaseⅠ, the effect of His-PEAc3-GFP was the most obviously. The activation effect of His-PEAc3-GFP on myosin Mg-ATPase activity was very obviously, reaching above five times than that of the blank control. The study shows that, the fusion of His and GFP could promote the correct expression and folding of PEAc3 in prokaryotic system, the fusion of GFP did not affect the biochemical characteristics of PEAc3 in vitro.The PEAc3 gene was cloned and expressed in E. coli. in this paper, and some biochemical properties of PEAc3 fusion with GFP were analysised, which was important for understanding higher plants actin isoforms deeply and studying the structure and function of actin. By GFP fusion expression system in this paper, we could not only gain abundant active plant actin isoforms for the relative research in vitro, but also study actins easily in vitro using the green fluorescent of GFP, and lay the foundation for the study of the dynamic changes of polymerization and depolymerization of actin iso forms in vivo.