Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 81 ·No. 8 ·1984-04-00 ·Pages 2285-9

Lo KM, Jones SS, Hackett NR, Khorana HG

Abstract

To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have introduced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr-185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (Xho I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA duplexes corresponding to the modified native and mutant restriction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rapid, efficient, and general method for purification of the synthetic oligonucleotides and of DNA fragments was developed.

Authors & Affiliations

4 authors, click to expand affiliations / ORCID

Lo K M

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

Jones S S

Hackett N R

Khorana H G

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Article Info

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abbr.

Proc Natl Acad Sci U S A

ISSN

0027-8424

DOI

10.1073/pnas.81.8.2285

Published

1984-04-00

Pages

2285-9

Language

English

Region

United States

NLM ID

7505876

PMCID

PMC345043

External Links

PubMed source DOI: 10.1073/pnas.81.8.2285 PMC Full Text View journal details

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Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons.

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