Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins.The current methods that applied in protein-protein interaction,such as co-immunoprecipitation and pull down etc.,often cause plenty of working time due to the burdensome cloning and puri-fication procedures.Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E.coli system.We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease,two cleavage site F and two multiple cloning sites that flanking cleavage sites.The target proteins(for example:protein A and protein B)were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His6 tag.PPV NIa protease carried out intracellular cleavage along expression,then led to the separation of polyprotein components,therefore,the interaction between protein A-protein B can be detected through one-step purification and analysis.Negative control for protein B was brought into this system for monitoring interaction specificity.We successfully employed this system to prove two cases of reported protien-protein interaction:RHA2a/ANAC and FTA/FTB.In conclusion,a convenient and efficient system has been successfully developed for detecting protein-protein interaction.
Plants are known to be efficient hosts for the production of mammalian therapeutic proteins.However,plants produce complex N-glycans bearingβ1,2-xylose and coreα1,3-fucose residues,which are absent in mammals.The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy.In this study,we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L.cv Bright Yellow 2(BY2),which is a well-established cell line widely used for the expression of therapeutic proteins.The expression of the endogenous xylosyltranferase(XylT)and fucosyltransferase(FucT)was downregulated by using RNA interference(RNAi)strategy.The xylosylated and core fucosylated N-glycans were significantly,but not completely,reduced in the glycoengineered lines.However,these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype.Therefore,this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.
Extreme environments such as salt stress often increase the reactive oxygen species(ROS) levels(Abogadallah,2010; Miller et al.,2010),which cause protein denaturation and damage(Kelsen et al.,2008).In eukaryotic cells,the endoplasmic reticulum(ER) lumen is highly oxidative environment, which help newly synthesized proteins form intermolecular or intramolecular disulfide bonds between their cysteine residues. However,the ROS must be regulated according to the folding load to protect the cell from the consequences of oxidative folding.One way to control ROS level is to increase the expression level of ROS scavenger(Molteni et al.,2004); another way is to activate unfolded protein response(UPR),
