Random changes of amino acid residues with expected frequency by saturated point mutagenesis

Seong Joong Kim, Hweon Park, Jeong Kook Kim, Jae Yung Lee, Kwangseog Ahn, MuHyeon Choe, Yong Jin Choi, Joon Kim

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The yeast transcriptional activator protein, Gcn4p from Saccharomyces cerevisiae binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control. A new random saturation mutagenesis method was developed to isolate Gcn4p derivatives with only one or two mutations in the DNA binding domain without using radioactive isotope. This will be used to identify the amino acids of Gcn4p involved in protein-protein interactions. Saturation mutagenesis in the DNA binding domain of Gcn4p was performed using spiked degenerate oligonucleotides containing randomized codon bases designed specifically for only one or two base changes in the mutagenized area. These oligonucleotides were synthesized to have two flanking restriction enzyme sites for cloning to the appropriate vector. The 3′ ends were mutually primed after hybridization via the palindromic sequences of the restriction enzyme sites. These molecules were then converted to double stranded DNA upon treatment with DNA polymerase. Here, a library collection of 100,680 in an altered Gcn4p pool was generated by cloning a mixed-base oligonucleotide in the place of the sequence coding for the DNA binding domains. The quality of the library was examined by DNA sequencing and found to be in good agreement with the expected statistical values. Calculated mutation frequency was 66% of mutant nucleotide rate and actual sequencing data revealed 68% mutant nucleotide rates from the sequenced library. Thus, among 21 mutants, 16 had one point mutations and 5 had two point mutations. This approach appears to be an effective and general tool for creating proteins with one or two amino acid change(s) in their molecules.

Original languageEnglish
Pages (from-to)232-235
Number of pages4
JournalMolecules and Cells
Volume10
Issue number2
Publication statusPublished - 2000 Apr 30

Fingerprint

Oligonucleotides
Mutagenesis
Libraries
Point Mutation
Amino Acids
Organism Cloning
DNA
Nucleotides
Saccharomyces cerevisiae Proteins
Proteins
Mutation Rate
DNA-Directed DNA Polymerase
Enzymes
DNA Sequence Analysis
Codon
Radioisotopes
Yeasts
Mutation
Genes
Therapeutics

Keywords

  • Mutually Primed Synthesis
  • Random Saturation Mutagenesis
  • Spiked Degenerate Oligonucleotide (Oligo)

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Random changes of amino acid residues with expected frequency by saturated point mutagenesis. / Kim, Seong Joong; Park, Hweon; Kim, Jeong Kook; Lee, Jae Yung; Ahn, Kwangseog; Choe, MuHyeon; Choi, Yong Jin; Kim, Joon.

In: Molecules and Cells, Vol. 10, No. 2, 30.04.2000, p. 232-235.

Research output: Contribution to journalArticle

Kim, Seong Joong ; Park, Hweon ; Kim, Jeong Kook ; Lee, Jae Yung ; Ahn, Kwangseog ; Choe, MuHyeon ; Choi, Yong Jin ; Kim, Joon. / Random changes of amino acid residues with expected frequency by saturated point mutagenesis. In: Molecules and Cells. 2000 ; Vol. 10, No. 2. pp. 232-235.
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