Use of a two-hybrid system to identify mutations in Max that confer increased affinity for Myc.

Abstract:

:A yeast two-hybrid system was used to identify mutants of Max that exhibit an increased affinity for Myc. Truncated forms of the Max helix-loop-helix/leucine zipper motif (HLH/Zip) were first expressed in a two- hybrid system in which the bait protein was the HLH/Zip motif of Myc. Deletion of amino acids both amino-terminal and carboxy-terminal to the leucine zipper of Max reduced Myc/Max heterodimer formation as evidenced by a 160-fold reduction in the expression of the lacZ gene. A library of partially randomized sequences encoding this minimal leucine zipper of Max was then screened using the two-hybrid system. Mutant forms of the Max leucine zipper were identified whose affinities for Myc, as measured by beta-galactosidase activity in yeast lysates, were from 8- to 200-fold greater than the wild-type Max zipper. These Max mutants were shown to interact specifically with Myc and not with wild-type Max. Of 29 mutants analyzed, all had a unique amino acid sequence. This result illustrates the value of a genetic screen in the identification of a collection of mutant forms of the Max leucine zipper whose structures would not have been predicted based on principles of structure-based design.

journal_name

DNA Cell Biol

journal_title

DNA and cell biology

authors

Wang H,Van Den Bergh F,Spencer E,Wilcox K,Herman T

doi

10.1089/dna.1997.16.1277

subject

Has Abstract

pub_date

1997-11-01 00:00:00

pages

1277-88

issue

11

eissn

1044-5498

issn

1557-7430

journal_volume

16

pub_type

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