Expression and purification of recombinant Physcomitrella patens DEK1-C2L proteins. Analysis of the phospholipid binding capacity of DEK1-C2L.
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Calpains are large group of ubiquitous and highly modular calcium dependent cysteine proteases that mainly are located in the cytoplasm of the cells and usually their activity is associated with binding to the phospholipids of intra cellular membranes. Calpains play critical role in regulating important physiologic functions in vast majority of different kinds of organisms, through performing limited cleavages of their substrates. Malfunction or dysfunction of calpains can cause serious disorders. The CysPc domain is the catalytic core domain of the calpain family and all proteins with a CysPc domain are considered as calpains. DEK1 is the only calpain family member that has been discovered in land pant so far. DEK1 structure is highly conserved among land plants through half billion years of evolution. This conservation of DEK1 indicates very important functions for this enzyme in land plants. Though, the exact function of DEK1 is not clear. Researches on DEK1 function have revealed that a functional dek1 gene is essential for maintaining the epidermal cell layer in developing tissues. Null mutation of dek1 causes abortion of embryo growth in land plants. Recent studies indicate a critical role of dek1 the in orientation of the correct cell division plane in the moss Physcomitrella patens. DEK1 is composed of a large transmembrane domain at the N-terminus that leads to the catalytic core domain of calpain family (CysPc) through a hydrophilic arm, and a C2L domain at the C-terminus. Limited experimental data concerning DEK1-C2L function is available, but significant level of conservation in amino acid sequences was detected between DEK1-C2L and animal calpain C2L domain indicating functional similarities. The C2L domain of animal calpain regulates enzyme activity via orchestrating Ca2+ and membrane binding. The work reported here is divided into two sub-projects. In the first part recombinant P. patents DEK1-C2L protein was expressed in Escherichia coli BL21 cells using the expression vector pET302 and the protein was purified by affinity chromatography. In the second part of the experiment, the phospholipid binding ability of the P. patents DEK1-C2L protein was investigated using size exclusion chromatography spin column technique. The work presented here suggests that the C2L domain of P. patens DEK1 bind to liposomes and this ability increases in presence of Ca2+.
Mastergradsoppgave i næringsrettet bioteknologi, Avdeling for lærerutdanning og naturvitenskap, Høgskolen i Hedmark, 2014. Master of applied and commercial biotechnology.