Title: Cloning and expression vectors and systems

Abstract

Vectors for cloning, maintaining and expressing a wide range of coding sequences in inducible T7 expression systems in Escherichia coli expression hosts are disclosed herein. Target genes that can be stably maintained and expressed include those that specify proteins that are highly toxic to the host cell. Different configurations of vectors and expression hosts provide different rates of transcription and translation of target genes and therefore different rates of accumulation of target proteins. Methods for cloning by asymmetric ligation and co-expression of more than one target protein in a single vector are also disclosed, as are variants of BL21(DE3) having lower basal transcription by T7 RNA polymerase.

Inventors:

Studier, F. William

Issue Date:

Tue Apr 04 00:00:00 EDT 2023

Research Org.:

Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1998257

Patent Number(s):

11618899

Application Number:

16/077,014

Assignee:

Brookhaven Science Associates, LLC (Upton, NY)

Patent Classifications (CPCs):

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C - CHEMISTRY C12 - BIOCHEMISTRY C12Y - ENZYMES

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C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C12N15/11 - DNA or RNA fragments
C12N15/63 - Introduction of foreign genetic material using vectors
C12N15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation
C12N15/67 - General methods for enhancing the expression
C12N15/70 - Vectors or expression systems specially adapted for E. coli
C12N9/1247 - {DNA-directed RNA polymerase

C - CHEMISTRY C12 - BIOCHEMISTRY C12Y - ENZYMES

C12Y207/07006 - DNA-directed RNA polymerase

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DOE Contract Number:  

SC0012704

Resource Type:

Patent

Resource Relation:

Patent File Date: 02/08/2017

Country of Publication:

United States

Language:

English