A plasmid was constructed that directs expression of the TL-DNA gene 4 protein in E. coli. The different steps of the construction were as follows : i) a region of gene 4 encoding the amino-terminal portion of the protein was fused in frame to DNA encoding an enzymatically active carboxy-terminal fragment of β-galactosidase. The hybrid gene was poorly expressed from the upstream λ PLpromoter carried by the vector. ii) in order to generate an efficient procaryotic ribosome binding site, a DNA fragment carrying the λ PRpromoter with the nearby Shine-Dalgarno (SD) sequence of gene cro was placed in front of the gene 4-lacZ fusion. A recombinant plasmid, termed pGV793, that expressed efficiently a fused protein 4-β-galactosidase was identified among the Lac+clones. DNA sequencing analysis showed that pGV793 carried a hybrid ribosome binding site composed of the cro SD sequence, a five bp sequence and the ATG codon of gene 4. Plasmid pGV793 directed the synthesis of three polypeptides of molecular weight 132 Kd, 126 Kd and 122 Kd that carried β-galactosidase antigenic determinants. The largest polypeptide had the expected size for the hybrid protein. The fusion proteins which accounted for about 0.5% of the total cellular proteins were purified by immunoadsorption using anti-β-galactosidase antiserum. iii) the complete gene 4 coding sequence was reconstituted, with the λ PRpromoter in place. The resulting pGV822 plasmid expressed a polypeptide whose molecular weight 27 Kd corresponded to the expected size for the gene 4 product. The pI was about 7. © 1984.
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