US 5,856,090 A · DNA-methylase linking reaction

Abstract

The activity of sequence-specific DNA binder proteins, such as DNA methylases, provides a method of obtaining a covalent linkage between a nucleic acid segment and a polypeptide determinant encoded by the nucleic acid segment. The polypeptide determinant is expressed as a fusion protein together with the DNA methylase, which binds in vivo to a cytidine suicide analog when present in a nucleotide sequence. A plasmid suitable for use in this linkage reaction can comprise: (1) a gene fusion construct including a gene encoding a DNA methylase and a gene encoding a polypeptide determinant; (2) a promoter for transcription of the gene fusion construct as messenger RNA; and (3) a methylase conjugation element linked to the gene fusion sequence, the methylase conjugation element including a methylase binding site having at least one copy of a nucleotide sequence including a cytidine suicide analog capable of irreversibly binding the DNA methylase. The plasmid can form a plasmid-polypeptide determinant conjugate. The plasmids and methods of the present invention are useful for in vitro evolution of proteins.

Claims51

Dependent claims

A plasmid comprising:

(a) a gene fusion construct including a gene encoding a DNA methylase and a gene encoding a polypeptide determinant covalently joined, either directly or through a linker, the gene fusion construct encoding a gene fusion product including a cytosine (C-5) DNA methylase having a methyltransferase activity and a polypeptide determinant covalently joined thereto, either directly or through a peptide linker;
(b) a promoter operatively linked to the gene fusion construct for promoting transcription of the gene fusion construct as messenger RNA;
and
(c) a methylase conjugation element linked to the gene fusion construct either directly or through an intervening sequence, the methylase conjugation element including a methylase binding site having at least one copy of a nucleotide sequence including a cytidine suicide analog capable of irreversibly binding the cytosine (C-5) DNA methylase within the gene fusion product wherein said methylase binding site nucleotide sequence is a methylase substrate for said cytosine (C-5) DNA methylase.

1 more element

A plasmid comprising:

(a) a gene fusion construct including a DNA methylase gene and a polypeptide determinant gene covalently joined thereto, the DNA methylase gene covalently joined contiguously in-frame to the polypeptide determinant gene with a linking orientation selected from the group consisting of a first orientation in which the 5'-terminus of the DNA methylase gene is covalently joined to the 3'-terminus of the polypeptide determinant gene and a second orientation in which the 3'-terminus of the DNA methylase gene is covalently joined to the 5'-terminus of the polypeptide determinant gene, the DNA methylase gene and the polypeptide determinant gene being joined through a linker for linking the DNA methylase gene to the polypeptide determinant gene, the linker encoding from 1 to 20 amino acids, the DNA methylase gene being selected from the group consisting of genes coding for Aqu I, Eag I, Eco 72 I, Hga I-1, Hga I-2, Hha I, Hpa II, Msp I, Nae I, Sss I, EcoR II, Hae III, NgoP II, Fnu DI, Fnu DII, Nla III, ScrF I, Sin I, Sso II, BsuF I, NgoM I, and Arabt, the gene fusion construct encoding a gene fusion product including a cytosine (C-5) DNA methylase having a methyltransferase activity and a polypeptide determinant covalently joined thereto;
(b) a prokaryotic promoter operatively linked to the gene fusion for promoting transcription of the gene fusion as messenger RNA, wherein the promoter promotes transcription in Escherichia coli;
and
(c) a methylase conjugation element linked to the gene fusion construct, the methylase conjugation element including from 1 to 50 copies of a methylase binding site, the methylase binding site having a nucleotide sequence that is a substrate for the methyltransferase activity of the cytosine (C-5) DNA methylase, the nucleotide sequence including a cytidine suicide analogue selected from the group consisting of 2'-deoxy-5-fluorocytidine, 2'-deoxy-5-azacytidine, and 2-pyrimidinone-1-.beta.-D-2-deoxyriboside wherein said methylase binding site nucleotide sequence is a methylase substrate for said cytosine (C-5) DNA methylase.

1 more element

A plasmid-polypeptide determinant conjugate comprising a plasmid and a gene fusion product covalently conjugated to the plasmid via a pyrimidine moiety of a cytidine suicide analogue, the plasmid including:

(a) a gene fusion construct including a DNA methylase gene and a polypeptide determinant gene covalently joined thereto, the gene fusion construct encoding a gene fusion product including a cytosine (C-5) DNA methylase having a methyltransferase activity and a polypeptide determinant covalently joined thereto;
(b) a promoter operatively linked to the gene fusion construct for promoting transcription of the gene fusion construct as messenger RNA;
and
(c) a methylase conjugation element linked to the gene fusion construct, the methylase conjugation element including a methylase binding site having a nucleotide sequence including a cytidine suicide analogue which irreversibly binds the cytosine (C-5) DNA methylase of the gene fusion product and having a methylation specificity for the cytosine (C-5) DNA methylase wherein said methylase binding site nucleotide sequence is a methylase substrate for said cytosine (C-5) DNA methylase.

1 more element

A library comprising a plurality of plasmid-polypeptide determinant conjugates, each plasmid-polypeptide determinant conjugate including:

(a) an encoding plasmid molecule including:
(i) a gene fusion construct including a DNA methylase gene and a polypeptide determinant gene covalently joined thereto, the DNA methylase gene covalently joined contiguously in-frame to the polypeptide determinant gene with a linking orientation selected from the group consisting of a first linking orientation in which the 5'-terminus of the DNA methylase gene is covalently joined to the 3'-terminus of the polypeptide determinant gene and a second linking orientation in which the 3'-terminus of the DNA methylase is covalently joined to the 5'-terminus of the polypeptide determinant gene, the DNA methylase gene selected from the group consisting of genes coding for Aqu I, Eag I, Eco 72 I, Hga I-1, Hga I-2, Hha I, Hpa II, Msp I, Nae I, Sss I, EcoR II, Hae III, NgoP II, Fnu DI, Fnu DII, Nla III, ScrF I, Sin I, Sso II, BsuF I, NgoM I, and Arabt, the gene fusion construct encoding a gene fusion product including a cytosine (C-5) DNA methylase having a methyltransferase activity and a polypeptide determinant covalently joined thereto;
(ii) a promoter operatively linked to the gene fusion construct for promoting transcription of the gene fusion construct as messenger RNA;
and
(iii) a methylase conjugation element linked to the gene fusion construct, the methylase conjugation element including a methylase binding site which serves as a substrate for the DNA methylase encoded by the DNA methylase gene, the methylase conjugation element having 1 to 50 copies of the methylase binding site, each methylase binding site including a nucleotide sequence including a cytidine suicide analogue selected from the group consisting of 2'-deoxy-5-fluorocytidine, 2'-deoxy-5-azacytidine, and 2'-pyrimidinone-1-.beta.-D-2-deoxyriboside wherein said methylase binding site nucleotide sequence is a methylase substrate for said cytosine (C-5) DNA methylase;
and
(b) said encoded gene fusion product covalently bound to said analogue.

4 more elements

A method for obtaining a nucleic acid segment encoding a polypeptide, the polypeptide having at least one property altered from a wild-type polypeptide, comprising the steps of:

(a) obtaining a nucleic acid segment encoding a wild-type polypeptide;
(b) subjecting the nucleic acid segment encoding the wild-type polypeptide to random mutagenesis to generate a plurality of mutagenized nucleic acid segments;
(c) constructing a library of plasmids from the plurality of mutagenized nucleic acid segments, each plasmid within the library of plasmids including:
(1) a gene fusion construct including a DNA methylase gene and one of the plurality of mutagenized nucleic acid sequences covalently joined thereto, the gene fusion construct encoding a gene fusion product including a cytosine (C-5) DNA methylase having a methyltransferase activity and a polypeptide determinant covalently joined thereto;
(2) a promoter operatively linked to the gene fusion construct for promoting transcription of the gene fusion construct as messenger RNA;
and
(3) a methylase conjugation element linked to the gene fusion construct, the methylase conjugation element including a methylase binding site having a nucleotide sequence including a cytidine suicide analog which irreversibly binds to the cytosine (C-5) DNA methylase of the gene fusion product wherein said methylase binding site nucleotide sequence is a methylase substrate for said cytosine (C-5) DNA methylase;
(d) causing expression of the gene fusion product from each plasmid of the library of plasmids;
(e) forming an irreversible joining of the gene fusion product to the plasmid encoding the gene fusion product for each plasmid of the library of plasmids, thereby forming a library of plasmid-polypeptide determinant conjugates;
(f) isolating a plasmid-polypeptide determinant conjugate from the library of plasmid-polypeptide determinant conjugates that binds a predetermined target moiety in a manner such that only plasmid-polypeptide determinant conjugates that have a property altered when compared to the wild-type polypeptide bind the target moiety;
(g) deproteinizing the plasmid-polypeptide determinant conjugate obtained in step (f) to form a purified plasmid encoding a polypeptide determinant having an altered property;
and
(h) isolating from the purified plasmid of step (g) a nucleic acid segment that encodes a polypeptide determinant having at least one property altered when compared with the wild-type polypeptide determinant.

10 more elements

A method for obtaining a nucleic acid segment encoding a polypeptide, the polypeptide having at least one property altered from a wild-type polypeptide, comprising the steps of:

(a) obtaining a nucleic acid segment encoding the wild-type polypeptide;
(b) subjecting the nucleic acid segment encoding the wild-type polypeptide to random mutagenesis to generate a plurality of mutagenized nucleic acid segments;
(c) constructing a library of plasmids from the plurality of mutagenized nucleic acid segments, each plasmid within the library of plasmids including:
(i) a gene fusion construct including a DNA methylase gene and one of the plurality of mutagenized nucleic acid sequences covalently joined thereto, the gene fusion construct encoding a gene fusion product including a cytosine (C-5) DNA methylase having a methyltransferase activity and a polypeptide determinant covalently joined thereto;
(ii) a promoter operatively linked to the gene fusion construct for promoting transcription of the gene fusion construct as messenger RNA;
and
(iii) a methylase conjugation element linked to the gene fusion construct, the methylase conjugation element including a methylase binding site having a nucleotide sequence including a cytidine suicide analog which irreversibly binds to the cytosine (C-5) DNA methylase of the gene fusion product wherein said methylase binding site nucleotide sequence is a methylase substrate for said cytosine (C-5) DNA methylase;
(d) causing expression of the gene fusion product from each plasmid of the library of plasmids;
(e) forming an irreversible joining of the gene fusion product to the plasmid encoding the gene fusion product for each plasmid of the library of plasmids, thereby forming a library of plasmid-polypeptide determinant conjugates;
(f) isolating a plasmid-polypeptide determinant conjugate from the library of plasmid-polypeptide determinant conjugates that binds a predetermined target moiety in a manner such that only plasmid-polypeptide determinant conjugates that have a property altered when compared to the wild-type polypeptide bind the target moiety;
(g) isolating from the purified plasmid of step (f) a nueleic acid segment that encodes a polypeptide determinant having at least one property altered when compared with the wild-type polypeptide determinant by amplifying the nucleic acid segment encoding the polypeptide determinant with the at least one altered property by a sequence-specific primer-based amplification method employing at least two primers.

8 more elements

(a) a gene fusion construct including a DNA methylase gene and a polypeptide determinant gene covalently joined thereto, the gene fusion construct encoding said gene fusion product including a cytosine (C-5) DNA methylase having a methyltransferase activity and a polypeptide determinant covalently joined thereto;
(b) a promoter operatively linked to the gene fusion construct for promoting transcription of the gene fusion construct as messenger RNA;
and
(c) a framework region including at least one methylase binding site having a nucleotide sequence including a cytidine suicide analogue which irreversibly binds the cytosine (C-5) DNA methylase of the gene fusion product and having a methylation specificity for the cytosine (C-5) DNA methylase wherein said methylase binding site nucleotide sequence is a methylase substrate for said cytosine (C-5) DNA methylase.

1 more element

Dependencies are inferred from the claim text.

DNA-methylase linking reaction

Life of this patent

DNA-methylase linking reaction

Life of this patent

Abstract

Claims51