Each vector is provided with an optional cmyc-tag for easy identification of expressed proteins using commercially available monoclonal antibodies (C-MYC info on the Biocompare.com site ). C-terminal to the c-myc tag a six histidine His-tag was added for simplified purification of your protein of interest using Ni-NTA affinity purification columns (His Tag info on the Biocompare.com site). The use of these tagging options requires that you clone the C-terminus of your protein in frame with the frame of the tag using either the NotI or BglII restriction sites.

Vector information
By default genes or gene fragments without pre-pro targeting peptides will express in the cytoplasm and this basic vector will help to achieve just that. It does provide the option of adding a tag to your protein of interest. The vector is also your choice if you want to express your gene with its own native targeting information.

Secreted expression is important for proper formation of disulphide bonds and the glycosylation, and is the first step in the sorting of proteins to the ER, Golgi, transport vesicles, the vacuole and the peroxisomes. The default pathway is secretion out of the cell into the cell wall and apoplast. Various sorting and retention signals determine the actual protein destination (see ImpactVector1.3). The vector can be used to change the targeting of the mature part of a protein to a secreted destination. The signal peptide used in this construct was derived from sea anemone equistatin (Outchkourov et al. 2003, in Planta and Plant Physiology).

By adding the well known KDEL retention or sorting signal to vector ImpactVector1.2 we created vector ImpactVector1.3. With this vector proteins are retained in the ER and do not go downstream into Golgi. ER retention has a practical advantage in that it has been reported to improve expression levels 5-fold or more. The main reason for this appears to be that the ER contains lower concentrations and/or different proteases responsible for post translational degradation of expressed proteins (Outchkourov et al. 2003)

Targeting to the chloroplast brings a protein into a completely different cellular environment capable of different metabolic and catabolic functions. ImpactVector1.4 vector uses the secretion signal of the Chrysanthemum morifolium small subunit protein to deliver your protein into the chloroplast stroma. The protein is fused to the first 11 amino acids of the rubisco protein in order to allow proper processing of the signal peptide (Wong et al., Plant Molecular Biology 20: 81-93 (1992)). The signal peptide contains a natural intron from the RbcS gene.

Targeting to mitochondria brings a protein to a completely different subcellular environment capable of different metabolic and catabolic functions. The ImpactVector1.5 vector uses the yeast CoxIV secretion signal which was shown to deliver proteins in the mitochondrial matrix. The protein is fused to the first 4 amino acids of the yeast CoxIV protein in order to allow proper processing of the signal peptide (Kohler et al. Plant J 11: 613-621 (1997)).