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The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence.

Hofbauer A, Peters J, Arcalis E, Rademacher T, Lampel J, Eudes F, Vitale A, Stoger E - Front Bioeng Biotechnol (2014)

Bottom Line: Endogenous PBs are derived from the endoplasmic reticulum (ER).The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain.Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences , Vienna , Austria.

ABSTRACT
Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

No MeSH data available.


Related in: MedlinePlus

(A) The N-terminal sequence of mature 27 kDa γ-zein, which has been shown to trigger PB formation. The sequence comprises two N-terminal cysteine residues (red) followed by an amphipathic repeat region and another four cysteine residues. The sequence present in construct ΔSP-DsCRS is boxed in orange. (B) Schematic overview of the expression constructs. SP, signal peptide for entry into the ER; TP, plastid-targeting peptide; DsZein, fluorescent marker protein plus γ-zein(4–93); CRS, cysteine-rich sequence (residues 51–93); HR, hydrophobic repeat sequence (PPPVRL)3; HIS, polyhistidine tag.
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Figure 1: (A) The N-terminal sequence of mature 27 kDa γ-zein, which has been shown to trigger PB formation. The sequence comprises two N-terminal cysteine residues (red) followed by an amphipathic repeat region and another four cysteine residues. The sequence present in construct ΔSP-DsCRS is boxed in orange. (B) Schematic overview of the expression constructs. SP, signal peptide for entry into the ER; TP, plastid-targeting peptide; DsZein, fluorescent marker protein plus γ-zein(4–93); CRS, cysteine-rich sequence (residues 51–93); HR, hydrophobic repeat sequence (PPPVRL)3; HIS, polyhistidine tag.

Mentions: All cloning steps were carried out with the binary vector pTRA, a derivative of pPAM (GenBank AY027531). Tetrameric DsRed (Jach et al., 2001) was joined via a (GGGS)2 linker to residues 4–93 of the mature 27 kDa γ-zein protein (lacking the signal peptide) in the same way as described for the PB-forming phaseolin fusion construct zeolin (Mainieri et al., 2004) to generate our basic vector ΔSP-DsZein. In construct SP-DsZein, a plant codon-optimized signal peptide sequence derived from a murine antibody (SP) was added at the N-terminus to direct the protein into the secretory pathway. In construct TP-DsZein, the 69-amino-acid plastid transit peptide sequence from the barley GBSSI gene (accession no. AF486514) was added instead. A peptide analysis using ChloroP prediction program (http://www.cbs.dtu.dk/services/ChloroP/) identified a potential chloroplast targeting peptide of 43 residues (cTP score of 0.460) in the N-terminal part of the mature 27 kDa γ-zein protein. This sequence comprising residues 51–93 of the γ-zein sequence was fused to DsRed via the (GGGS)2 linker to test the predicted function of this peptide. The resulting construct was termed ΔSP-DsCRS. For the control construct SP-DsHR, we used three repeats of the sequence PPPVRL fused to DsRed. This sequence is similar to the repeat domain of the zein sequence and does not include cysteine residues. A His6 tag was added to the C-terminus of all constructs as an additional means for detection. The synthetic fusion sequences were inserted into the pTRA-vector between the Tobacco etch virus (TEV) 5′-untranslated region and the Cauliflower mosaic virus (CaMV) 35S terminator (Figure 1). The expression construct was thus placed under the control of the CaMV 35S promoter with duplicated transcriptional enhancer.


The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence.

Hofbauer A, Peters J, Arcalis E, Rademacher T, Lampel J, Eudes F, Vitale A, Stoger E - Front Bioeng Biotechnol (2014)

(A) The N-terminal sequence of mature 27 kDa γ-zein, which has been shown to trigger PB formation. The sequence comprises two N-terminal cysteine residues (red) followed by an amphipathic repeat region and another four cysteine residues. The sequence present in construct ΔSP-DsCRS is boxed in orange. (B) Schematic overview of the expression constructs. SP, signal peptide for entry into the ER; TP, plastid-targeting peptide; DsZein, fluorescent marker protein plus γ-zein(4–93); CRS, cysteine-rich sequence (residues 51–93); HR, hydrophobic repeat sequence (PPPVRL)3; HIS, polyhistidine tag.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263181&req=5

Figure 1: (A) The N-terminal sequence of mature 27 kDa γ-zein, which has been shown to trigger PB formation. The sequence comprises two N-terminal cysteine residues (red) followed by an amphipathic repeat region and another four cysteine residues. The sequence present in construct ΔSP-DsCRS is boxed in orange. (B) Schematic overview of the expression constructs. SP, signal peptide for entry into the ER; TP, plastid-targeting peptide; DsZein, fluorescent marker protein plus γ-zein(4–93); CRS, cysteine-rich sequence (residues 51–93); HR, hydrophobic repeat sequence (PPPVRL)3; HIS, polyhistidine tag.
Mentions: All cloning steps were carried out with the binary vector pTRA, a derivative of pPAM (GenBank AY027531). Tetrameric DsRed (Jach et al., 2001) was joined via a (GGGS)2 linker to residues 4–93 of the mature 27 kDa γ-zein protein (lacking the signal peptide) in the same way as described for the PB-forming phaseolin fusion construct zeolin (Mainieri et al., 2004) to generate our basic vector ΔSP-DsZein. In construct SP-DsZein, a plant codon-optimized signal peptide sequence derived from a murine antibody (SP) was added at the N-terminus to direct the protein into the secretory pathway. In construct TP-DsZein, the 69-amino-acid plastid transit peptide sequence from the barley GBSSI gene (accession no. AF486514) was added instead. A peptide analysis using ChloroP prediction program (http://www.cbs.dtu.dk/services/ChloroP/) identified a potential chloroplast targeting peptide of 43 residues (cTP score of 0.460) in the N-terminal part of the mature 27 kDa γ-zein protein. This sequence comprising residues 51–93 of the γ-zein sequence was fused to DsRed via the (GGGS)2 linker to test the predicted function of this peptide. The resulting construct was termed ΔSP-DsCRS. For the control construct SP-DsHR, we used three repeats of the sequence PPPVRL fused to DsRed. This sequence is similar to the repeat domain of the zein sequence and does not include cysteine residues. A His6 tag was added to the C-terminus of all constructs as an additional means for detection. The synthetic fusion sequences were inserted into the pTRA-vector between the Tobacco etch virus (TEV) 5′-untranslated region and the Cauliflower mosaic virus (CaMV) 35S terminator (Figure 1). The expression construct was thus placed under the control of the CaMV 35S promoter with duplicated transcriptional enhancer.

Bottom Line: Endogenous PBs are derived from the endoplasmic reticulum (ER).The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain.Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences , Vienna , Austria.

ABSTRACT
Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.

No MeSH data available.


Related in: MedlinePlus