Expression of a functional recombinant fusion protein via the directional sub-cloning of an E.coli derived tyrosine phosphatase gene (wzb) into a pT5(6H)CFP mutant expression vector.
Abstract: Application of fluorescent fusion proteins to the field of expression and interaction proteomics as a means of dynamic imaging proteins in vivo has allowed for rapid advancements in biotechnology research. Production of such proteins first involves the insertion of a given protein-coding gene transcriptionally in-frame with a fluorophore sequence under the control of a single promoter and terminator. With reference to this experiment, successful BamHI and PstI digestion of both pHSG-WZB donor (coding for wzb tyrosine phosphatase) and pT5(6H)CFP recipient vectors allowed for ligation formation of pT5WZB(6H)CFP (coding for desired fluorescent fusion protein). Transformation into competent E.coli and plating against TE buffer and pHSG-WZB controls experimentally disputed theoretical expectations as ampicillin resistance (50725 cfu/mL) and fluorescence (93%) along with kanamycin resistance (66800 cfu/mL) and non-fluorescence were displayed, suggesting ineffective fragment ligation. Using effectively ligated transformants, screening for positive clones via plasmid extraction and PstI and XhoI digestion displayed two expected fragments; pT5 (experimental: 2339 bp) and cfp-wzb (experimental: 1198 bp). SDS-PAGE analysis of auto-induced positive clone cell lysate revealed the expression of the cfp-wzb fusion protein (experimental: 44.3 kDa). Using a hexahistidine tag, successful fusion protein purification was achieved via nickel affinity chromatography and confirmed by SDS-PAGE. Finally, para-nitrophenyl phosphate assay of tyrosine phosphatase activity allowed for determination of wzb kinetic properties such as Km (6.34 mM) and Vmax (0.0644 µmol/min/mg). Gaining an experimental competency in formation, detection and use of specific fusion proteins will ultimately allow for the appreciation of their importance in research involving protein localisation and/or trafficking.
Introduction: Generation of fluorescent recombinant fusion proteins involves the upstream or downstream insertion of a protein-coding gene of interest that is transcriptionally in-frame with a fluorophore-coding sequence not endogenously present within the culturing organism. Investigation into the functionality surrounding fluorophore-tagged fusion proteins has been a focal point of concerted research in recent decades due to its extensive experimental potential in the visualisation of gene and protein expression in vivo. Given the variable nature of this process, its applications are widespread and may involve visualising; spindle assembly and cell division in real-time mitosis, protein-protein interactions, intracellular transport of proteins within the endomembrane system and cellular compartmentalisation in plants (Ehrenberg 2008; Hanson & Köhler 2001). With reference to this particular experiment, cyan mutant of green fluorescent protein coding sequence was transcribed in-frame with wzb tyrosine phosphatase gene of interest along with a hexahistidine purification tag to ultimately generate a fluorescent and enzymatically functional recombinant.
Originally derived from Aequorea victoria, green fluorescent protein has been utilised extensively in molecular biology as a means of dynamic imaging of proteins in vivo. The induction of mutation at the 65th and/or 66th codon position have been shown to shift the excitation and emission peaks of the associated polypeptide chromophore to give rise to phenotypic variants e.g. Y66W mutation produces cyan fluroescent protein (Tsein 1998). Fluorescence of these proteins is the result of energy transfer from aequorin Ca2+-activated photoprotein following ultraviolet excitation (Barnum 2005). When mRNA translation of the fusion protein coding sequence occurs, the autocatalytic (host and exogenous substrate independent)...
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