Directed evolution of protein in mammalian cells

Details Directed evolution of protein in mammalian cells Directed evolution of protein in mammalian cells

2002-05-20

2004-04-20

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S006120, C536S023100, C530S350000

Reexamination Certificate

active

06723537

ABSTRACT:

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
1. Field of the Invention
The present invention relates to directed protein evolution in mammalian cells and improved mutants of Discosoma sp. red fluorescent proteins.
2. Background of the Invention
Red fluorescent protein has been isolated from a Discosoma sp. and sequenced (see, e.g., Matz et al., Nature Biotech. 17:969-973 (1999), Gross et al.,
Proc. Nat'l Acad. Sci. USA
97:11990-11995 (2000)). A variant with humanized codons has also been engineered (Clontech, “DSREDT™”). The crystal structure of red fluorescent protein has been elucidated, which demonstrated that red fluorescent protein is a tetrameric protein (Wall et al.,
Nat. Struc. Biol.
7:1089 (2000); Yarbrough et al.,
Proc. Nat'l Acad. Sci USA
16:462-467 (2000)).
Red fluorescent protein (RFP) and DsRED, as well as other fluorescent proteins such as YFP, or GFP from
Aequorea Victoria, Renilla reniformis, Renilla muelleri,
and
Ptilosarcus gurneyi,
are useful are reporter molecules for a variety of bioassays, including those that use FACS as a selection mechanism (see, e.g., Tsein,
Nature Biotechnology
17:956 (1999); Tsein,
Ann. Rev. Biochem.
6:509-544 (1998); Heim et al.,
Nature
373:663-664 (1995); Heim et al.,
Proc. Nat'l Acad. Sci. USA
91:1250 (1994); Prasher et al.,
Gene
111:229 (1992); Prasher et al.,
Trends in Genetics
11:320 (1995); Chalfie et al.,
Science
263:802 (1994); and WO 95/21191). However, brighter, faster folding, and higher expressing variants would be useful.
Such variants can be made, e.g., using methods of gene shuffling and mutagenesis (see, e.g., U.S. Pat. No. 5,811,238; WO 00/73433; WO 00/22115; WO 99/41369; WO 01/04287; WO 00/46344; WO 99/45143, WO 99/41368; and Ichiro et al.,
Protein Science
8:731-740 (1999)). However, the use of such methods for production of variant proteins such as Discosoma red fluorescent protein variants is not always successful (see, e.g., Baird et al.,
Proc. Nat'l Acad. Sci. USA
97:11984-11989 (2000)). Novel methods of making such variants would therefore be useful.
SUMMARY OF THE INVENTION
The present invention therefore provides variants of Discosoma red fluorescent protein that have been generated using directed molecular evolution in mammalian cells. The variants of the invention have greatly improved brightness, expression, and/or folding kinetics as compared to wild type or a codon optimized variant. The present invention also provides novel methods of directed protein evolution in mammalian cells using retroviral gene transfer and FACS sorting. Such methods can be used to provide improved variants of fluorescent proteins such as Discosoma red fluorescent protein and fluorescent proteins from other sources, such as
Aequorea victoria, Renilla reniformis, Renilla muelleri,
and
Ptilosarcus gurneyi.
In one aspect, the present invention provides an isolated Discosoma red fluorescent protein, the protein comprising an amino acid sequence as shown in
FIG. 1
with one or more point mutations at an amino acid position selected from the group consisting of N24, F125, K164, and M183.
In one embodiment, the protein comprises two, three, or four point mutations at an amino acid position selected from the group consisting of N24, F125, K164, and M183.
In one embodiment, the point mutation at amino acid position N24 is a serine, arginine, or histidine substitution. In another embodiment, the point mutation at amino acid position F125 is a leucine or valine substitution. In another embodiment, the point mutation at amino acid position K164 is a methionine substitution. In another embodiment, the point mutation at amino acid position M183 is a lysine or threonine substitution.
In one embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a leucine or valine substitution at amino acid position F125 and a lysine substitution at amino acid position M183. In another embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a leucine substitution at amino acid position F125 and a lysine substitution at amino acid position M183. In another embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a valine substitution at amino acid position F125 and a lysine substitution at amino acid position M183.
In one embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a leucine or valine substitution at amino acid position F125 and a serine, arginine, or histidine substitution at amino acid position N24. In another embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a leucine substitution at amino acid position F125 and a serine substitution at amino acid position N24.
In one embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a leucine or valine substitution at amino acid position F125, a serine, arginine, or histidine substitution at amino acid position N24, and a lysine substitution at amino acid position M183. In another embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a leucine substitution at amino acid position F125, a serine substitution at amino acid position N24, and a lysine substitution at amino acid position M183.
In one embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a methionine substitution at amino acid position K164.
In one embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a leucine substitution at amino acid position F125.
In one embodiment, the protein further comprises one or more point mutations at an amino acid position selected from the group consisting of K93, R18, K139, E149, and D170. In another embodiment, the point mutation at amino acid position K93 is an arginine substitution. In another embodiment, the point mutation at amino acid position R18 is a histidine substitution. In another embodiment, the point mutation at amino acid position E149 is an aspartic acid substitution. In another embodiment, the point mutation at amino acid position D170 is a glycine substitution.
In one embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a leucine substitution at amino acid position F125, a serine substitution at amino acid position N24, a lysine substitution at amino acid position M183, and a histidine substitution at amino acid position R18.
In one embodiment, the protein comprises an amino acid sequence as shown in
FIG. 1
with a leucine substitution at amino acid position F125, a aspartic acid substitution at amino acid position E149, and a glycine substitution at amino acid position D170.
In one aspect, the present invention provides a Discosoma red fluorescent protein that is a fusion protein.
In another aspect, the present invention provides a nucleic acid encoding the Discosoma red fluorescent protein of the invention. In one embodiment, the nucleic acid is codon-optimized for mammalian expression. In another embodiment, the nucleic acid encodes a fusion protein.
In another aspect, the present invention provides a vector comprising a nucleic acid encoding the Discosoma red fluorescent protein of the invention. In one embodiment, the vector is a retroviral vector.
In another aspect, the present invention provides a host cell comprising the vector of the invention.
In another aspect, the present invention provides a retroviral cDNA expression library comprising a nucleic acid encoding the Discosoma red fluorescent protein.
In another aspect, the present invention provides a method of making a protein variant, the method comprising the steps of: (i) mutating a selected nucleotide sequence encoding a fluorescent protein; (ii) cloning the mutated sequences into an expression vector; (iii) transfecting mammalian cells with the expression vector; and (iv) identifying the variants.
In one embodiment, the protein is a fluorescent protein and variants are identified by FACS analysis. In another embodiment, the selecte

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