Inducible gene expression composition for using eukaryotic pol-2 promoter-driven transcription in prokaryotes and the applications thereof

• US 9,783,811 B2
• Filed: 09/30/2014
• Issued: 10/10/2017
• Est. Priority Date: 08/12/2011
• Status: Active Grant

• Current Assignee: Mello Biotechnology, Inc.
• Original Assignee: Mello Biotechnology, Inc.
• Inventors: Lin, Shi-Lung, Chang, Donald C.
• Primary Examiner(s): Nguyen, Quang
• Application Number:
  US14/502,608
• Publication Number:
  US 20150064771A1
• Time in Patent Office: 1,106 Days
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 1/38 Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound C12N1/34 takes precedence
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

Eukaryotic protein-coding messenger RNAs and non-coding microRNAs are naturally transcribed by type II RNA polymerases (pol-2) but not prokaryotic RNA polymerases. As a result, current eukaryotic RNA and protein production is performed either using eukaryotic pol-2 promoters in hybridomas or mammalian cells or using prokaryotic promoters in bacterial cells. However, because prokaryotic RNA transcription tends to be error-prone, frequent mutation is a big problem. Also, growing hybridomas or mammalian cells is relatively laborious and costly. To overcome these problems, the present invention provides a novel inducible composition and method for producing eukaryotic RNAs and/or their related peptides/proteins directly using eukaryotic pol-2 promoter-driven gene expression in fast growing bacteria, without the need of changing to prokaryotic promoters or growing hybridomas/mammalian cells. The RNAs and peptides/proteins so obtained can be used to develop drugs, cure diseases, treat tumors/cancers, produce pluripotent stem (iPS) cells, enhance wound healing, and make foods.

Claims

1. A composition for regulating eukaryotic elongation factor-1 alpha (EF1alpha) promoter-driven gene expression in prokaryotes, comprising:

(a) at least a chemical agent capable of inducing or enhancing eukaryotic EF1alpha promoter-driven gene expression in prokaryotic cells, wherein said chemical agent containing a structure of 3-morpholinopropane-1-sulfonic acid (MOPS); and

(b) a plurality of prokaryotic cells, said plurality of prokaryotic cells containing at least a gene mediated by a eukaryotic EF1alpha promoter-driven expression mechanism and wherein said gene encodes at least a hairpin-like structure in its 5′

-untranslated region (5′

-UTR) for being expressed in prokaryotes;

wherein (a) and (b) are mixed together under a condition to induce the eukaryotic EF1alpha promoter-driven gene expression of said gene in said prokaryotic cells;

wherein said condition is in Luria-Bertani (LB) broth.

2. The composition as defined in claim 1, wherein each one of said prokaryotic cells has at least an inducible gene expression composition.

3. The composition as defined in claim 2, wherein said gene is located in a plasmid vector containing at least a eukaryotic EF1alpha promoter.

4. The composition as defined in claim 1, wherein said gene encodes at least a non-coding RNA and a protein-coding RNA.

5. The composition as defined in claim 4, wherein said non-coding RNA is a small hairpin RNA.

6. The composition as defined in claim 4, wherein said protein-coding RNA further encodes a red fluorescent protein (RGFP).

7. The composition as defined in claim 4, wherein said protein-coding RNA contains 30% to 100% homology to a cDNA sequence of a eukaryotic gene.

8. The composition as defined in claim 4, wherein said non-coding RNA or said protein-coding RNA is isolatable from said prokaryotes.

9. The composition as defined in claim 1, wherein said gene encodes at least a protein.

10. The composition as defined in claim 4, wherein a protein transcribed from said protein-coding RNA contains at least a peptide sequence.

11. The composition as defined in claim 10, wherein said protein or said peptide is isolatable from said prokaryotes.

12. The composition as defined in claim 4, wherein a protein transcribed from said protein-coding RNA is an enzyme.

13. The composition as defined in claim 1, wherein said prokaryotic cells are bacterial cells.

14. The composition as defined in claim 1, wherein said prokaryotic cells are Escherichia coli (E. coli).

15. The composition as defined in claim 1, wherein said chemical agent is a transcription inducer for inducing RNA expression of said gene encoding at least the hairpin-like structure in said prokaryotic cells.

16. The composition as defined in claim 1, wherein said chemical agent in said condition has a v/v concentration of 0.01% to 1%.

17. The composition as defined in claim 4, wherein said non-coding RNA is useful for pharmaceutical or therapeutic application.

18. The composition as defined in claim 4, wherein said protein-coding RNA is useful for pharmaceutical or therapeutic application.

19. The composition as defined in claim 4, wherein said non-coding RNA is a precursor of microRNA.

20. The composition as defined in claim 19, wherein said precursor of microRNA is a precursor of miR-302.

21. The composition as defined in claim 4, wherein a protein transcribed from said protein-coding RNA is useful for pharmaceutical or therapeutic application.

22. The composition as defined in claim 17, wherein said pharmaceutical or therapeutic application is cancer therapy and disease treatment.