Enhanced in vitro protein synthesis
OwnerKangseok Lee, Stanley N. Coheninventors
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Abstract
Poly G tails prolong mRNA chemical and functional half life in E. coli cell extracts and dramatically increased RNA-dependent protein synthesis in vitro. The effect of polyguanylation on mRNA functional half life, as measured by the ability of CAT transcripts to produce biochemically-active protein in vitro, was four- to six-fold greater than the effect on chemical half life. Addition of a few nucleotides 5′ to the bacteriophage T7 promoter markedly enhanced transcription of linear PCR-generated DNA molecules by T7 RNA polymerase. Collectively a novel approach is provided for efficient in vitro protein synthesis that bypasses the need for cloned DNA templates to obtain the products of translational open reading frames.
Claims23
A method for enhanced synthesis of a polypeptide in an expression system, the method comprising:
- stabilizing mRNA encoding said polypeptide by insertion of a stabilizing sequence at the 3′ non-coding region of said mRNA,
- translating said mRNA into its encoded protein in an in vitro expression system;
- wherein said stabilized mRNA has an increased functional half-life and provides for enhanced protein synthesis.
A method of enhancing transcription from a linear DNA with a phage specific promoter, the method comprising:
- enhancing transcription by a phage specific RNA polymerase by insertion of a transcription enhancing sequences 5′ to said promoter.
A method for synthesis of a targeted polypeptide, the method comprising:
- amplifying a targeted polynucleotide sequence with a 5′ and a 3′ primer, wherein said 5′ primer comprises a T7 promoter and a transcription enhancing sequence 5′ to said promoter;
- and wherein said 3′ primer comprises an mRNA stabilization sequence;
- and wherein said primers specifically amplify said targeted polynucleotide sequence;
- transcribing the amplification product into mRNA with T7 RNA polymerase;
- translating said mRNA in vitro;
- wherein said targeted polypeptide is synthesized.
A method for determining the identity of the protein product of a targeted polynucleotide, the method comprising:
- amplifying a targeted polynucleotide sequence, wherein said targeted polynucleotide sequence is present in a complex mixture of sequences, with a 5′ and a 3′ primer, wherein said 5′ primer comprises a T7 promoter and a transcription enhancing sequence 5′ to said promoter;
- and wherein said 3′ primer comprises an mRNA stabilization sequence;
- and wherein said primers specifically amplify said targeted polynucleotide sequence;
- transcribing the amplification product into mRNA with T7 RNA polymerase;
- translating said mRNA in vitro;
- wherein said targeted polypeptide is synthesized;
- combining said synthesized polypeptide with a cellular lysate;
- analyzing said cellular lysate by gel electrophoresis;
- wherein the position of said synthesized identifies the protein product of the targeted polynucleotide.
A method for synthesizing polypeptides encoded by uncloned genomic open reading frames, the method comprising:
- amplifying a targeted polynucleotide sequence, wherein said targeted polynucleotide sequence is an uncloned genomic open reading frame, with a 5′ and a 3′ primer, wherein said 5′ primer comprises a T7 promoter and a transcription enhancing sequence 5′ to said promoter;
- and wherein said 3′ primer comprises an mRNA stabilization sequence;
- and wherein said primers specifically amplify said targeted polynucleotide sequence;
- and wherein said primers insert an epitope tag into said genomic open reading frame;
- transcribing the amplification product into mRNA with T7 RNA polymerase;
- translating said mRNA in vitro;
- wherein said targeted polypeptide is synthesized;
- isolating said synthesized polypeptide by binding to said epitope tag.
Dependencies are inferred from the claim text.