Vicilin with carboxy‐terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic plants

Abstract

SummaryGene constructs were designed to test the effect of the endoplasmic reticulum (El?)-targeting signal, KDEL, on the level of accumulation of a foreign protein in transgenic plants. The gene for the pea seed protein vicilin was modified by the addition of a sequence coding for this tetrapeptide at its carboxyl terminus. The altered gene was placed under the control of a CaMV 35s promoter and its expression in the leaves of both tobacco and lucerne (alfalfa) was compared with that of an equivalent vicilin construct lacking the KDELcoding sequence. The presence of the ER-targeting signal led to a greatly enhanced accumulation of the heterologous protein. In lucerne and tobacco leaves, the level of vicilin-KDEL protein was 20 and 100 times greater than that of the unmodified vicilin, respectively. These differences in expression level could not be explained by corresponding differences in the steadystate levels or the translatability of the mRNAs. However, when the stability of vicilin and vicilin-KDEL proteins was compared in their respective transgenic hosts, unmodified vicilin was found to be degraded with a half-life of 4.5 h while vicilin-KDEL was much more stable with a half-life of more than 48 h. lmmunogold labelling of leaf tissues from transgenic lucerne and tobacco showed the presence of vicilin associated with large aggregates within the ER lumen of vicilin-KDEL plants. No such aggregates were detected in transgenic plants expressing wild-type vicilin.It is concluded that the carboxy-terminal KDEL caused the retention of the modified vicilin in the ER, and that this retention led to the increased stability and higher level of accumulation of vicilin-KDEL in leaves of transgenic plants.

Developmental changes in abundance of the VSPβ protein following nuclear transformation of maize with the Soybean vspβ cDNA

“…Accounting for the differences in total protein applied to the gel (less soybean total protein was loaded), the VSPβ protein was estimated to have accumulated to 0.5 % of the total soluble protein. This is similar to the highest level of seed storage protein accumulation observed with the ectopic expression of zein [8,9], but remains less than the 1% minimal expression level predicted by Wandelt et al [11] to be needed to directly alter the nutritional quality of the leaves. Although the 0.5% of total soluble protein was too low to alter nutritional value, detection of VSPβ in 45-3-1 allowed monitoring of VSPβ level in leaves and stems during plant development.…”

“…Transgenic expression from the CaMV 35S promoter in tobacco resulted in the formation of these protein bodies containing the zein within vegetative tissues [8,9]. Alternatively, "short-circuiting" the protein-targeting route by addition of an ER retention signal to the storage-protein coding region also increased protein accumulation up to 100× [5,6,10,11]. …”

Influence of Elastin-Like Polypeptide and Hydrophobin on Recombinant Hemagglutinin Accumulations in Transgenic Tobacco Plants

“…The H5 protein was recombinantly expressed alone (H5) or as a fusion with hydrophobin (H5-HFBI) or ELP (H5-ELP) under the control of both the CaMV 35S and USP promoters (Figure 1). Each recombinant protein contained the c-myc tag for downstream detection by Western blot, a His tag for purification by immobilized metal ion chromatography (IMAC) and the KDEL motif at the C-terminal end to retain the protein in the endoplasmic reticulum (ER) [43]. The functionality of all binary vectors was validated by assaying the expression of recombinant proteins in transiently transformed N. benthamiana leaves and confirmed by Western blot (data not shown).…”

“…N -linked glycans are then transported and modified further in the GA from cis to medial and trans cisternae to form typical plant glycans with the addition of xylose and fucose to the core glycans. In this study, the KDEL motif was used to retain recombinant proteins in the ER [43]. The N -linked glycan profiles of these recombinant proteins showed that leaf-derived H5 and H5-HFBI predominantly possess OMT glycans (70–72%) that supposedly matured in the ER compartment and 27–30% CT glycans with typical plant glycans (GnGnX and GnGnF) (Table 2).…”

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates

“…Both wild type and codon-optimized pfa genes were cloned into pCaMterX binary vector [17] under the control of double 35S promoter [29] and Nos terminator [30]. Tobacco secretory signal peptide Pr1b [31] was fused to the N-terminal of the gene and StrepII tag [32] and the ER retrieval tetrapeptide KDEL [33] were fused to the C-terminus of the gene…”