What question did this study set out to answer?

To establish an effective purification method for the labile, biologically active replication initiator protein of plasmid R6K using gene fusion and subunit interaction strategies.

November 1, 1983Proceedings of the National Academy of SciencesOpen Access

Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.

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Authors

JGJoseph GerminoRutgers, The State University of New Jersey JGJohn G. GrayCanadian Food Inspection Agency HCHarry CharbonneauPurdue University West Lafayette

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Implication

Experimental study demonstrates successful isolation of active plasmid R6K replication initiator protein via lacZ fusions and subunit interactions, suggesting a general strategy for labile...

Key Points

To establish an effective purification method for the labile, biologically active replication initiator protein of plasmid R6K using gene fusion and subunit interaction strategies.
Fused the cistron of the plasmid R6K initiator protein near its COOH-terminus to the Escherichia coli lacZ cistron at the ninth codon from the NH2-terminus.
Purified the resulting hybrid protein and co-associated native subunits using beta-galactosidase-specific affinity chromatography.
Conducted NH2-terminal amino acid sequence analysis on the purified heteropolymer to confirm protein identity against nucleotide sequence predictions.
The initiator-lacZ fusion yielded a stable protein exhibiting dual activities: beta-galactoside hydrolysis and replication initiation in vivo and in vitro.
Beta-galactosidase-specific affinity chromatography achieved rapid purification of the hybrid fusion protein to near homogeneity.
Subunit interactions within the initiator's polymeric structure enabled the co-purification of native, nonfused initiator protein, confirming identical NH2-terminal sequences.

Cite This Study

Germino et al. (1983) studied this question.

synapsesocial.com/papers/6a756ff7e239d27fbe0333e9 https://doi.org/10.1073/pnas.80.22.6848

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Implication

Experimental study demonstrates successful isolation of active plasmid R6K replication initiator protein via lacZ fusions and subunit interactions, suggesting a general strategy for labile...

Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.

Discussion

Member takes

Implication

Key Points

Cite This Study

Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K.

Discussion

Member takes

Implication

Key Points

Cite This Study

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