Methods and compositions for protein engineering
AssigneePrimrose Bio, Inc.
InventorsHelge ZIELER, Dongxin Karen XU, Sabrina BAFFERT, Patrik ENGSTROM, Shawn WALSH
Life of this patent
Estimating the term…
Abstract
The present disclosure provides compositions and methods for creating screening systems using RNA polymerase promoters and sequences encoding RNA polymerases for isolating genes encoding variant RNA polymerases with altered or improved properties related to in vitro transcription, or for isolating RNA polymerase promoter sequences, or for isolating untranslated sequences contributing to higher rates of protein synthesis from a nucleic acid molecule encoding a polypeptide or protein.
Claims25
A screening system for isolating nucleic acid sequences encoding RNA polymerases with altered properties compared to a parental RNA polymerase, containing one or more nucleic acid templates comprising:
- (a) An RNA polymerase promoter;
- and
- (b) A sequence encoding an RNA polymerase that recognizes the promoter in part (a), placed downstream of the promoter in part (a) such that the encoded RNA polymerase can transcribe the sequences encoding it.
A process for improving RNA polymerase activity comprising:
- (a) Creating a collection of in vitro compartments containing a mixture of:
- i) a collection of nucleic acid templates encoding RNA polymerases with altered properties compared to a parental RNA polymerase which recognizes a first promoter present on the nucleic acid templates;
- ii) an initiating RNA polymerase that recognizes a second promoter present on the nucleic acid templates of (i);
- iii) proteins, nucleic acids and other reaction components necessary for in vitro transcription and translation;
- (b) Incubating the mixture of step (a) under conditions in which the RNA polymerases encoded in the nucleic acid templates produce RNA transcripts encoding RNA polymerases;
- (c) Isolating the RNA transcripts produced in step (b) and processing the isolated RNA transcripts to regenerate nucleic acid templates encoding RNA polymerases;
- and
- (d) Repeating steps (a)-(c) to enrich for nucleic acid template molecules encoding RNA polymerases with altered activity or properties compared to a parental RNA polymerase.
A nucleic acid molecule comprising:
- (a) Two promoter sequences recognized by RNA polymerases;
- and
- (b) A sequence encoding an RNA polymerase that recognizes one of the promoters in part (a),
- wherein each of the two promoter sequences in part (a) directs transcription of the sequence encoding the RNA polymerase of (b).
A process for isolating promoter sequences recognized by an RNA polymerase, comprising:
- (a) Creating a collection of in vitro compartments containing a mixture of:
- i) An initiating RNA polymerase;
- ii) a collection of nucleic acid templates encoding a second RNA polymerase distinct from the initiating RNA polymerase, a first promoter recognized by the encoded RNA polymerase, and diverse sequences upstream of the RNA polymerase coding sequence that serve as the source of promoter elements for the initiating RNA polymerase of (i);
- iii) proteins, nucleic acids and other reaction components necessary for in vitro transcription and translation;
- (b) Incubating the mixture of step (a) under conditions in which the RNA polymerases encoded in the nucleic acid templates produce RNA transcripts encoding RNA polymerases;
- (c) Isolating the RNA transcripts produced in step (b) and processing the isolated RNA transcripts to regenerate nucleic acid templates encoding RNA polymerases;
- and
- (d) Repeating steps (a)-(c) to enrich for nucleic acid template molecules encoding promoter sequences recognized by the initiating RNA polymerase.
A process for improving RNA polymerase coding sequences comprising:
- (a) Creating a collection of in vitro compartments containing a mixture of:
- i) a collection of nucleic acid templates of different sequences encoding the same RNA polymerase which recognizes a first promoter present on the nucleic acid templates;
- ii) an initiating RNA polymerase that recognizes a second promoter present on the nucleic acid templates of (i);
- iii) proteins, nucleic acids and other reaction components necessary for in vitro transcription and translation;
- (b) Incubating the mixture of step (a) under conditions in which the RNA polymerases encoded in the nucleic acid templates produce RNA transcripts encoding RNA polymerases;
- (c) Isolating the RNA transcripts produced in step (b) and processing the isolated RNA transcripts to regenerate nucleic acid templates encoding RNA polymerases;
- and
- (d) Repeating steps (a)-(c) to enrich for nucleic acid template molecules encoding RNA polymerases transcribed or translated at higher levels than a specific reference sequence encoding the RNA polymerase.
Dependencies are inferred from the claim text.