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

• US 9,637,747 B2
• Filed: 10/29/2014
• Issued: 05/02/2017
• Est. Priority Date: 08/12/2011
• Status: Active Grant

• Current Assignee: Mello Biotechnology, Inc., Wjwu & Lynn Institute For Stem Cell Research
• Original Assignee: Mello Biotechnology, Inc.
• Inventors: Lin, Shi-Lung, Chang, Donald C.
• Primary Examiner(s): Nguyen, Quang
• Application Number:
  US14/527,439
• Publication Number:
  US 20150118734A1
• Time in Patent Office: 916 Days
• Field of Search: 435 712, 435471, 435476
• 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 method for inducing or enhancing eukaryotic EF1alpha promoter-driven gene expression in prokaryotes, comprising:

contacting a chemical agent, containing a structure of 3-morpholinopropane-1-sulfonic acid (MOPS) with said prokaryotes containing said eukaryotic EF1alpha promoter-driven gene, wherein said eukaryotic EF1alpha promoter-driven gene encodes at least a hairpin-like RNA structure in its 5′

-untranslated region (5′

-UTR).

2. The method as defined in claim 1, further comprising mixing said chemical agent with glycerin to enhance the sufficiency of eukaryotic EF1alpha promoter-driven gene expression.

3. The method as defined in claim 1, wherein said method is performed in a bacterial culturing medium.

4. The method as defined in claim 3, wherein said chemical agent has a v/v concentration of 0.01% to 1% in said bacterial culturing medium.

5. The method as defined in claim 3, wherein said bacterial culturing medium is Luria-Bertani (LB) broth.

6. The method as defined in claim 1, wherein said eukaryotic EF1alpha promoter-driven gene encodes at least a protein/peptide and non-coding RNA, which is useful for pharmaceutical or therapeutic application.

7. The method as defined in claim 6, wherein said non-coding RNA is small hairpin RNA useful for pharmaceutical or therapeutic application.

8. The method as defined in claim 6, wherein said non-coding RNA is a miR-302 homologue.

9. The method as defined in claim 6, wherein said protein or peptide is an enzyme useful for pharmaceutical or therapeutic application.

10. The method as defined in claim 1, wherein said prokaryotes are bacterial cells.

11. The method as defined in claim 10, wherein said bacterial prokaryotic cells are Escherichia coli (E. coli).

12. The method as defined in claim 1, wherein said eukaryotic EF1alpha promoter-driven gene is located in a plasmid vector.

13. The method as defined in claim 12, wherein said plasmid vector is pLenti-EF1alpha-RGFP-miR302.