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The Fusion Expression of a Pea Actin Isoform (PEAc3) and Green Fluorescent Protein (GFP) and the Biochemical Characteristics Analysis to the Fusion Protein

Author: LiuZuo
Tutor: ZhangShaoBin
School: Shenyang Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Plant actin isoforms Sequence analysis GFP fusion The prokaryotic expression Protein Purification
CLC: Q946.1
Type: Master's thesis
Year: 2010
Downloads: 42
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Abstract

Actin is ubiquitous in eukaryotes, under the control of various actin-binding protein involved in a variety of cellular activities, including cell division, endocytosis, the nucleation cell signaling, gravity sensor and top growth organelle movement. The existence of multiple isoforms of different actin isoform expression in plant cells by a multigene family encoding actin organization organ-specific play different functions in the process of plant growth and development. To better understand the function of specific actin isoform and its dynamic regulation GFP fusion technology peas muscle actin isoforms 3 (PEAc3) prokaryotic expression and purification, and some of its biochemical properties, mainly The results are as follows: the cloned pea actin protein isoforms PEAc3 gene by RT-PCR method, randomly selected seven kinds of other plant actin homology of 90% or more. Using DNAman (Ver6.0) bioinformatics software, Pea seed isoform between biological evolution tree, provide the basis for the study of eukaryotic molecular evolutionary relationship. Amino acid sequence based on the predicted PEAc3 the presumption PEAc3 protein MW 41.67kD pI of 5.17, and the soluble protein without the signal peptide sequence, exists in the form of a mature protein, actin-containing proteins typical three groups of sequences fingerprint speculated PEAc3 dimensional structure Figure with existing animal skeletal muscle actin is similar to the three-dimensional structure. In order to study the impact of gene fusion PEAc3 prokaryotic expression, were constructed of four prokaryotic expression the carrier PEAc3-pET30a the His-PEAc3 pET30a, of His-PEAc3-GFP-pET30a and His-of GFP-pET30a. DNAman software analysis of the protein expressed by the above carrier, the full length of 377 PEAc3 amino the Mw for 41.67kD, pI of 5.17; the His-PEAc3 total length of 427 amino acids, the Mw for 47.13kD, pI of 5.56; a His-GFP- The full-length PEAc3 of 675 amino acids, MW 74.74kD a pI of 5.81; His-GFP total length of 296 amino acids, MW 33.04kD a pI of 6.26. The carrier will be built by electroporation into E. coli BL21 induced expression conditions optimized: PEAc3 add optimal bacterial concentration of IPTG induction OD600 = 0.7, IPTG concentration of 0.05mmol / L, inducing expression time of 4h, the temperature was 25 ° C, expressed mainly in inclusion bodies in bacterial concentration; His-PEAc3 OD600 = 0.5 when you add IPTG was concentration O.lmmol / L induced expression time 3h, a temperature of 37 ° C. , expressed mainly in inclusion bodies; His-PEAc3-GFP at a the bacterium concentration of OD600 = 0.8 add IPTG, a concentration of 0.1 mmol / L, induced expression time of 4 h and a temperature of 25 ° C in the supernatant and inclusion body expression; His-GFP in bacterial concentration as the OD600 of = 0.5 when IPTG was added, the concentration of 0.05mmol / L induced expression time 4h, the temperature was 37 ° C, is expressed primarily in the supernatant. The above results show that, the GFP fusion certain role in promoting soluble expression in prokaryotic system for PEAc3. Refolding of urea-denatured nickel affinity chromatography, ion exchange chromatography, these four proteins were purified, and some of its biochemical characteristics. His-PEAc3-GFP polymerization kinetics and animal skeletal muscle actin closer comparison PEAc3 His-PEAc3 and His-PEAc3-GFP curve of polymerization and polymer critical concentration. DNase I activity was measured, PEAc3 His-PEAc3 and His-PEAc3-GFP DNase I inhibited His-PEAc3-GFP inhibitory effect is most obvious. His-PEAc3-GFP has the obvious effect activation of myosin Mg-ATPase activity, to blank five times. These studies show that the His and GFP fusion PEAc3 correct expression and folding in prokaryotic systems to promote the role of the GFP fusion did not affect PEAc3 vitro biochemical characteristics. The subject of a pea muscle actin isoforms PEAc3 gene cloning and expression, its GFP fusion protein biochemical characteristics were analyzed, and the results are important for the understanding of the higher plant actin isoforms body, exploring the actin structure and function has significance. GFP fusion expression not only can get a large number of active plant actin isoform, thus vitro experimental material, while taking advantage of the green fluorescence of GFP can be convenient in vitro research on the one hand, on the other hand to study the in vivo isoforms of actin polymerization depolymerization of dynamic changes in a certain basis.

The Fusion Expression of a Pea Actin Isoform (PEAc3) and Green Fluorescent Protein (GFP) and the Biochemical Characteristics Analysis to the Fusion Protein

Abstract

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