High level expression of proteins

• US 5,795,737 A
• Filed: 09/22/1995
• Issued: 08/18/1998
• Est. Priority Date: 09/19/1994
• Status: Expired due to Term

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Seed, Brian, Haas, Jurgen
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): LONGTON, ENRIQUE D
• Application Number:
  US08/532,390
• Time in Patent Office: 1,061 Days
• Field of Search: 536/23.5, 536/23.1, 435/320.1, 435/240.2, 435/252.3, 435/254.11, 435/240.4, 435/254.2, 435/183, 435/69.1
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/70503 Immunoglobulin superfamily
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2740/16122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

The invention features a synthetic gene encoding a protein normally expressed in a mammalian cell or eukaryotic cell wherein at least one non-preferred or less preferred codon in the natural gene encoding the mammalian protein has been replaced by a preferred codon encoding the same amino acid.

Claims

1. A synthetic gene encoding a green fluorescent protein, wherein at least one non-preferred or less preferred codon in the natural gene encoding said protein has been replaced by a preferred codon encoding the same amino acid, said preferred codons being selected from the group consisting of gcc, cgc, aac, gac, tgc, cag, ggc, cac, atc, ctg, aag, ccc, ttc, agc, acc, tac, and gtg, said less preferred codons being selected from the group consisting of ggg, att, ctc, tcc, and gtc, said non-preferred codons being all codons other than said preferred codons and said less preferred codons, wherein said synthetic gene permits the expression of said green fluorescent protein in a mammalian host cell at a level which is at least 110% of that expressed by said natural gene in an in vitro mammalian cell culture system under identical conditions.

2. The synthetic gene of claim 1 wherein said synthetic gene permits the expression of said green fluorescent protein in a mammalian host cell at a level which is at least 150% of that expressed by said natural gene in an in vitro mammalian cell culture system under identical conditions.

3. The synthetic gene of claim 1 wherein said synthetic gene permits the expression of said green fluorescent protein in a mammalian host cell at a level which is at least 200% of that expressed by said natural gene in an in vitro mammalian cell culture system under identical conditions.

4. The synthetic gene of claim 1 wherein said synthetic gene permits the expression of said green fluorescent protein in a mammalian host cell at a level which is at least 500% of that expressed by said natural gene in an in vitro mammalian cell culture system under identical conditions.

5. The synthetic gene of claim 1 wherein said synthetic gene permits the expression of said green fluorescent protein in a mammalian host cell at a level which is at least ten times that expressed by said natural gene in an in vitro mammalian cell culture system under identical conditions.

6. The synthetic gene of claim 1 wherein at least 10% of the codons in said natural gene are non-preferred codons.

7. The synthetic gene of claim 6 wherein at least 50% of the codons in said natural gene are non-preferred codons.

8. The synthetic gene of claim 1 wherein at least 50% of the non-preferred codons and less preferred codons present in said natural gene have been replaced by preferred codons.

9. The synthetic gene of claim 1 wherein at least 90% of the non-preferred codons and less preferred codons present in said natural gene have been replaced by preferred codons.

11. The synthetic gene of claim 1 wherein said synthetic gene permits the expression of said green fluorescent protein in a mammalian host cell at a level which is at least 1000% of that expressed by said natural gene in an in vitro mammalian cell culture system under identical conditions.

12. The synthetic gene of claim 1 having the sequence depicted in FIG. 11 (SEQ ID NO:

40).

13. An expression plasmid comprising the synthetic gene of claim 1.

14. A mammalian cell-transfected with the expression plasmid of claim 13.

10. A method for preparing a synthetic gene encoding a green fluorescent protein, comprising identifying non-preferred and less preferred codons in the natural gene encoding said protein and replacing one or more of said non-preferred and less preferred codons with a preferred codon encoding the same amino acid as the replaced codon, said preferred codons being selected from the group consisting of gcc, cgc, aac, gac, tgc, cag, ggc, cac, atc, ctg, aag, ccc, ttc, agc, acc, tac, and gtg, said less preferred codons being selected from the group consisting of ggg, att, etc, tcc, and gtc, said non-preferred codons being all codons other than said preferred codons and said less preferred codons, wherein said synthetic gene permits the expression of said green fluorescent protein in a mammalian host cell at a level which is at least 110% of that expressed by said natural gene in an in vitro mammalian cell culture system under identical conditions.