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Methods and cells for expression of recombinant protein products under the transcriptional control of an inducible promoter

a technology of inducible promoter and recombinant protein, which is applied in the field of methods and cells for expression of recombinant protein products under the transcriptional control of inducible promoter, can solve the problems of low protein expression level of uninduced ara system, inability to modulate the expression of cloned genes, and inability to induce ara system protein expression, etc., to achieve efficient and economical production, increase the expression of recombinant protein products, and reduce the effect of ara

Inactive Publication Date: 2005-11-24
BETTER MARC D
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides improved methods for producing recombinant protein products using inducible promoters, such as the araB promoter. The invention involves genetically engineering bacterial host cells with deficiencies in active transport systems for inducers of the promoter. The invention offers advantages such as increased expression of the recombinant protein product, lower inhibition of host cell growth after induction, and direct and synchronous induction of expression in the host cells. The invention also provides a bacterial host cell that is deficient in one or more of the active transport systems for an inducer, such as L-arabinose, and contains an expression vector encoding a recombinant protein product under the transcriptional control of an inducible promoter. The recombinant protein products may include therapeutic, prophylactic, and diagnostic agents."

Problems solved by technology

For example, many recombinant products can be toxic to the expression host.
Additionally, the uninduced level of protein expression from the ara system is very low.
Thus, these references, including Siegele and Hu, supra, conclude that although araB vectors have the advantages of rapid regulation and low basal level expression compared to plasmids regulated by the lac repressor, the araB promoter is not well-suited to modulate the expression of cloned genes because of variances in arabinose uptake between cells.
However, a problem still facing the art is to generate directly regulated cell cultures in which the amount of protein expression in the cells is actually proportional to the amount of inducer (e.g., arabinose) present in culture medium.

Method used

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  • Methods and cells for expression of recombinant protein products under the transcriptional control of an inducible promoter
  • Methods and cells for expression of recombinant protein products under the transcriptional control of an inducible promoter
  • Methods and cells for expression of recombinant protein products under the transcriptional control of an inducible promoter

Examples

Experimental program
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Effect test

example 1

Construction of Arabinose Transport-Deficient Strains

[0039] Isogenic ΔaraFGH::kan and araE201 strains were constructed from E. coli W3110 (ATCC 27325) and E. coli E104 (deposited as ATCC 69009; ATCC 69008; ATCC 69101; ATCC 69102; ATCC 69103; ATCC 69104; ATCC 69331; ATCC 69332; ATCC 69333, each of which contains a gelonin-encoding plasmid). The resultant strains along with intermediate strains are shown in Table 1.

TABLE 1Arabinose Proficient and Deficient StrainsStrainParent or ReferenceRelevant GenotypeCW2553Horazdovsky and Hogg, 1989,ΔaraFGH::kan araE201Jour. of Bacter. 171: 3053-3059E104W3110 / See, Example 1Δ(araBC)768W3110ATCC 27325E220W3110 / PTA-1524AΔaraFGH::kanE221E104 / PTA-1525AΔaraFGH::kanE222W3310thyAE223E104thyAE224E222 / PTA-1526AaraE201E225E223 / PTA-1527AaraE201E226E224 / E228 / PTA-1528AΔaraFGH::kan araE201E227E229 / PTA-1529AΔaraFGH::kan araE201E228E220ΔaraFGH::kan thyAE229E221ΔaraFGH::kan thyA

ADeposited with the American Type Culture Collection (ATCC) on Mar. 21, 2000.

Constr...

example 2

SDS-PAGE and ELISA Analysis of Gelonin Expression in W3110, E220, E224 and E226 after Arabinose Induction

[0047] Four isogenic strain (W3110, E220, E224 and E226) were transformed with pING3825. Each cell line was grown to an OD600 of approximately 0.4, then aliquots of 2 mL of each culture were transferred to tubes supplemented with arabinose to 0, 0.01, 0.1 or 1%. Twenty eight tubes were set up, so that cells induced with 0, 0.01, 0.1 or 1% arabinose could be harvested at 4 hours post-induction and cells induced with 0.01, 0.1 and 1% arabinose could be harvested 18 hours post-induction. After the appropriate induction period of either 4 or 18 hours, cells were removed from the culture supernatant by centrifugation and each supernatant was filtered through a 0.2 μm filter before storage at 4° C. Ten microliters of each supernatant was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples from each supernatant were stained with Coomassie colloida...

example 3

Analysis of Cell Growth and Gelonin Expression in E104, E221, E225 and E227 after Arabinose Induction

[0052] Four Δ(araBD)768 bacterial cell lines (E104, E221, E225 and E227) each containing pING3825 were grown in TYE medium to an OD of approximately 0.4 and induced in 2.15 mL aliquots with arabinose to 0, 0.1, 1 and 10 mM (10 mM 0.15%; 1 mM 0.015%; 0.1 mM 0.0015%). At 4 hours post-induction, the OD600 of a 1:5 dilution of each culture was determined, then the culture was centrifuged to remove cells and the filtered supernatant was placed at −20° C. for subsequent evaluation by ELISA. Likewise, at 17 hours post-induction, the OD600 of a 1:10 dilution of each culture was determined, then the culture was centrifuged to remove cells and the filtered supernatant was placed at −20 C for subsequent evaluation by ELISA. Table 4 illustrates the culture OD600 for each sample.

TABLE 4Optical Density of E104, E221, E225 and E227 cultures inducedwith arabinoseStrainmM arabinoseOD (4 hours)OD (...

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Abstract

The present invention relates generally to improved methods for the expression of recombinant protein products under the transcriptional control of an inducible promoter, such as an araB promoter, in bacterial host cells that are deficient in one or more of the active transport systems for the inducer of the inducible promoter. The present invention also relates to improved bacterial host cells that are deficient in one or more of the active transport systems for an inducer of an inducible promoter, such as arabinose for an araB promoter, and contain an expression vector encoding a recombinant polypeptide under the transcriptional control of the inducible promoter, such as an araB promoter.

Description

[0001] The present invention relates generally to improved methods for the expression of recombinant protein products under the transcriptional control of an inducible promoter, such as the araB promoter, in bacterial host cells that are deficient in one or more of the active transport systems for an inducer. In the case of the araB promoter, the inducer is L-arabinose. The present invention also relates to improved bacterial host cells that are deficient in one or more of the active transport systems for an inducer, such as L-arabinose, and that contain an expression vector encoding a recombinant protein product under the transcriptional control of an inducible promoter, such as the araB promoter. BACKGROUND OF THE INVENTION [0002] Although many systems have been described for expression of recombinant proteins, including peptides and polypeptides, in microbial systems, most gene expression systems in gram negative bacteria such as Escherichia coli have relied exclusively on a limi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09C12N1/21C12N15/63C12N15/70C12P21/02
CPCC12N15/635C12P21/02C12N15/70
Inventor BETTER, MARC D.
Owner BETTER MARC D
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