Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli

Abstract

This paper reports the expression of an artificial functional polypeptide in bacteria. The gene of a designed 24-residue DDT-binding polypeptide (DBP) was inserted between the BamYQ and PsA cleavage sites of plasmid pUR291. The hybrid plasmid, pUR291-DBP, was cloned in Escherichia coli JM109. After induction by isopropyl-/3-D-thiogalactopyranoside a fusion protein was expressed in which DBP was linked to the COOH-tenninus of (3-galactosidase. DBP, which is stable to trypsin, was obtained by tryptic digestion of the fusion protein and subsequent fractionation of the tryptic peptides by reversed-phase h.p.l.c. Recombinant and chemically synthesized DBP showed identical chromatographic properties, amino add composition, and chymotryptic digestion patterns. Both the /3-galactosidase-DBP fusion and isolated recombinant DBP bound DDT. The fusion protein was 25 times as potent as the designed 24-residue DBP in activating a cytochrome P-450 model system using equimolar catalytic amounts of the two proteins. Key words: protein design/DDT-binding polypeptide/pUR291 hybrid plasmid/fusion protein/recombinant DDT-binding polypeptide could be efficiently expressed in a suitable organism, and (ii) if there was expression, the physicochemical and biological properties of the primary translation product (inevitably a fusion protein containing the DBP sequence) could be studied, and DDT binding and metabolism by a DBP + and a DBP~ strain of E. coli could be compared. Materials and methods Construction of the hybrid plasmid pUC8-DBPThe eight DNA fragments constituting the double-stranded DNA of the DBP gene (Figure 2, nucleotide sequences in boxes) for insertion between the BamHl and Pstl cleavage sites of die polylinker of plasmid pUC8 (Vieira and Messing, 1982) were synthesized by the solid phase phosphoramidite method (Beaucage and Caruthers, 1981) using protected deoxyribonucleotidemorpholino-methoxyphosphines (Dorper and Winnacker, 1983). After synthesis and complete deprotection they were purified by electrophoresis on preparative denaturing 16% polyacrylamide gels. The bands containing the target oligodeoxynucleotides were

Rapid partial degradation of DDT by a cytochrome P‐450 model system

“…The reaction time for each measurement was 6 h to bind DDT using secondary structure prediction and model building whereas /I-casein can be considered a natural DDTbinding protein [22,23]. The dissociation constants of the two protein-DDT complexes were 0.8 pM [24] and 55 pM [23] (Fig. 9).…”

“…The objective of the present investigation was to develop a model system for DDT degradation and to study the influence of DDT-binding proteins, in particular a designed 24 [4]. Aerobic reactions catalyzed by (inducible) cytochrome P-450 enzymes yield mainly DDA [his@-chloropheny1)acetic acid, Fig.…”

Fusion Proteins from Artificial and Natural Structural Modules

“…We then tried to construct a DBP-producing E. coli strain expressing the peptide from the recombinant hybrid plasmid pUC8-DBP [25] and able to remove DDT contaminations from the environment. However, despite its strongly hydrophobic character DBP was largely digested by intracellular proteases and therefore could not be detected immunologically using anti-DBP antiserum [25].…”

“…However, despite its strongly hydrophobic character DBP was largely digested by intracellular proteases and therefore could not be detected immunologically using anti-DBP antiserum [25]. DBP was much more stable intracellularly when produced as a fusion protein with ß-galactosidase.…”

“…DBP was much more stable intracellularly when produced as a fusion protein with ß-galactosidase. The order of the components of the corresponding 1050-residue construct expressed from the recombinant hybrid plasmid pUR291-DBP was as follows: ß-galactosidase (1-1021)-Arg-Gly-Ser-Lys-Arg-DBP(24 residues) [25].…”