Co-expression of a precursor and the mature protein of wheat ribulose-1,5-bisphosphate carboxylase small subunit from a single gene in Escherichia coli.
The cDNA encoding a precursor of wheat ribulose-1,5-bisphosphate carboxylase/oxygenase was inserted in-phase with prokaryotic expression elements in four different vectors. Five expression vectors encoding the small subunit precursors were cloned in Escherichia coli. None of these constructs expressed detectable amounts of the precursor protein, but all directed synthesis of the mature small subunit. The expression of the small subunit was a consequence of an independent, intragenic Shine-Dalgarno sequence optimally located upstream from an ATG specifying the first codon of the mature small subunit portion in the precursor transcript. Similar internal translation signals have been identified in the nuclear-encoded cDNAs of the small-subunit precursors of numerous higher plant genes. The 5' end of the wheat small-subunit precursor was linked with a consensus E. coli DNA sequence such that the modified gene encoded a partial hybrid precursor carrying four additional residues at its amino terminus. The resultant construct, pEI-W3, directed abundant synthesis of both the partially hybrid small-subunit precursor and the mature small subunit, constituting as much as 10% of the total bacterial protein. The bacterially synthesized small subunit precursor was purified to homogeneity. The authenticity of the recombinant protein was verified by its size, immunological properties, amino-terminal sequence, and amino acid composition.
Kaderbhai
M A
MA
Department of Biochemistry, University College of Wales, Aberystwyth, Dyfed, UK.
He
M Y
MY
Beechey
R B
RB
Kaderbhai
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010944:Plants / Q000201:enzymology; D014176:Protein Biosynthesis / Q000235:genetics; D012333:RNA, Messenger; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014908:Triticum / Q000201:enzymology*
D004792:Enzyme Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase