A General Method for Retrieving the Components of a Genetically Engineered Fusion Protein

Szoka, Paula; Schreiber, Alain B.; Chan, Hardy; Murthy, Janaki

doi:10.1089/dna.1986.5.11

Cited by 33 publications

(11 citation statements)

References 30 publications

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“…The enzyme is also apparently degraded during two-dimensional gel electrophoresis with the loss of Mr 15,000 because this procedure yielded a major protein of =30,000 Mr (6). This result can now be explained from the derived amino acid sequence, which contains two acid-labile aspartyl-proline bonds (18). Cleavage of these bonds at low pH during isoelectric focusing would give three fragments of Mr 28,000, 15,000, and 1000.…”

Section: Discussionmentioning

confidence: 96%

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

Lange

Hedden

Graebe

1994

Proc. Natl. Acad. Sci. U.S.A.

164

133

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In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in Agtll was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA2s and of GA53 to GA17, as well as the formation of the C,,-GAs, GA1, GA,, and GA20, from their respective aldehyde precursors, GA23, GA2,4, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted Mr (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases.The gibberellins (GAs) form a large group of diterpenoid natural products. Certain GAs function as hormones in plants, controlling many aspects of development, including stem extension, fruit set, and seed germination (1). The later steps of the GA biosynthetic pathway are catalyzed by soluble 2-oxoglutarate-dependent dioxygenases, which oxidize the GA skeleton at the 20, 3,8, and 2p3 positions (see Fig.

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Section: Discussionmentioning

confidence: 96%

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

Lange

Hedden

Graebe

1994

Proc. Natl. Acad. Sci. U.S.A.

164

133

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“…Clostripain Bennett et al (1984) (Szoka et al, 1986). Low pH treatment was performed in the presence of guanidinium chloride.…”

Section: Fusion Proteinsmentioning

confidence: 99%

The purification of eukaryotic polypeptides synthesized in Escherichia coli

Marston¹

1986

Biochemical Journal

858

379

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“…Finally, methods exist to separate a foreign protein from a vector after purification. In the case of MalE, the following methods seem appropriate: (a) cleavage at a cysteine after cyanylation since MalE does not contain any cysteine [39]; (b) cleavage at a dipeptide Asp-Pro by treatment with formic acid since MalE only contains one such dipeptide that could be removed by mutagenesis [40]; (c) cleavage to the tetrapeptide Ile-Glu-Gly-Arg by blood-clotting factor X, [41]; (d) cleavage to a pentapeptide Pro-Xaa-GlyPro-Yaa with collagenase [42]. However, in many applications, cleavage is not required.…”

Section: Comparison With Other Systemsmentioning

confidence: 99%

Production in Escherichia coli and one‐step purification of bifunctional hybrid proteins which bind maltose

Bedouelle

Duplay

1988

European Journal of Biochemistry

124

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Two enzymes, the secreted Staphylococcus aureus nuclease A and the Klenow fragment of the cytoplasmic Escherichia coli DNA polymerase I, were fused, at the genetic level, to MalE, the periplasmic maltose-binding protein of E. coli, or to a signal-sequence mutant. The hybrid proteins were synthesized in large amounts by E. coli under control of promoter malEp. The synthesis was repressed with glucose and could be totally switched off in a malT mutant strain. The hybrid between MalE and the nuclease was exported into the periplasmic space. Several criteria demonstrated that a fraction of the hybrid chains with the Klenow polymerase was exported to the periplasm in a signal-sequence-specific manner and ruled out the possibility of a membrane leakage. The hybrid with the Klenow polymerase was not exported and remained in the cytoplasm when carrying a tight signalsequence mutation in its MalE portion. The hybrid proteins were purified in one step by affinity chromatography on cross-linked amylose. Most of the hybrid chains in the periplasm but only a fraction of those in the other cell compartments had their MalE portion correctly folded. The nuclease and the Klenow polymerase had their full specific activities in the purified hybrids. The potential of MalE as a vector for the production, export and purification of desirable proteins in E. coli is discussed.

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A General Method for Retrieving the Components of a Genetically Engineered Fusion Protein

Cited by 33 publications

References 30 publications

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

The purification of eukaryotic polypeptides synthesized in Escherichia coli

Production in Escherichia coli and one‐step purification of bifunctional hybrid proteins which bind maltose

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