Targeting of a foreign protein to chloroplasts by fusion to the transit peptide of ribulose 1,5-bisphosphate carboxylase. Nature 313: 358-363

Nature (Impact Factor: 41.46). 01/1985; 313(6001):358-63. DOI: 10.1038/313358a0
Source: PubMed


Chimaeric genes can be constructed which fuse the transit peptide of a small subunit of the chloroplast-located ribulose 1,5-bisphosphate carboxylase with a bacterial protein. The fusion protein is translocated into chloroplasts and cleaved in a similar way to the small subunit polypeptide precursor.

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    • "Three cassettes that harboured a non-maize codon-optimized version (Chiu et al., 1996) were assembled under the control of either the 35S CaMV (Benfey and Chua, 1990), rice actin promoter , coupled with a 5¢-intron (Zhong et al., 1996) or the 1.7 kb maize C4 PepC promoter. (Yanagisawa and Izui, 1989) (ZmPpc1) The respective promoters were fused with translational enhancer element from the maize PPDK-A gene (Sheen, 1993), and GFP was targeted to plastids via the maize chloroplast RNA polymerase RpoTp transit peptide (Chang et al., 1999) for the 35S CaMV and rice actin cassettes, or the pea RBCS1 transit peptide (Van den Broeck et al., 1985; von Heijne et al., 1991) for the PEPC cassette. The non-codon-optimized GFP cassettes were subcloned into either the binary plasmid pPZP211 or pPZP212 (Hajdukiewicz et al., 1994), and the resultant vectors were referred to as pPTN343, pPTN372 and pPTN442, for the 35S, rice actin and PEPC promoters, respectively. "
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    • "To prove this hypothesis, a protein was designed that can be targeted to both the chloroplast and the peroxisome. It is well known that the N-terminal transit peptide is necessary and sufficient for targeting a protein to the chloroplast (Van den Broeck et al., 1985; Smeekens et al., 1986; Bassham et al., 1991; Bruce, 2000; Lee et al., 2002). In contrast, the C-terminal three amino acid residues, SKL, are necessary and sufficient for targeting a protein to the peroxisome (Nito et al., 2002; Sparkes and Baker, 2002). "
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    • "Even very subtle changes in the vicinity of the natural cleavage site of the Rubisco small subunit precursor can lead to diminished chloroplast uptake (Wasmann et al., 1988) and/or aberrant proteolytic processing (Robinson and Ellis, 1984, 1985). It is occasionally observed that chloroplast uptake of foreign proteins can be improved by including a small portion of the mature N terminus of the transit peptide donor in addition to the transit peptide and scissile bond (Schreier et al., 1985; Van den Broeck et al., 1985). However, this approach is still associated with a high degree of unpredictability that is inextricably linked to the passenger protein. "
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