Isolation and Characterization of Novel Green Fluorescent Proteins from Copepods
AssigneeBoard of Regents, The University of Texas System
InventorsMikhail V. Matz, Marguerite E. Hunt
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Abstract
The isolation and characterization of two protein isoforms collected green fluorescent copepods is described herein. The new Pontella mimocerami GFP-like isoforms pmimGFP1 and pmimGFP2 of the present invention are quick to mature and rapidly produce a fluorescent signal. The two isoforms are very similar in molar extinction coefficients (ME) with 105,000 M−1 cm−1 for pmimGFP1 and 103,000 M−1cm−1 for pmimGFP2, respectively. The relative brightness of these two new copepod GFP-like proteins is the highest measured for any isolated GFP-like protein.
Claims33
An isolated nucleic acid molecule comprising a nucleotide sequence that is at least 95% homologous to a SEQ ID NO.:
- 2 or to a SEQ ID NO.:
- 4.
An isolated protein molecule comprising an amino acid sequence, wherein the amino acid sequence is at least 95% identical to a SEQ ID NO.:
- 1 or to a SEQ ID NO.:
- 3.
An expression vector comprising one or more nucleic acid sequences, wherein the nucleic acid sequence encodes one or more functional proteins.
A host cell comprising a nucleic acid sequence wherein the nucleic acid sequence is at least 95% homologous to a SEQ ID NO.:
- 2 or to a SEQ ID NO.:
- 4, wherein the nucleic acid sequence encodes one or more functional proteins having an amino acid sequence that is at least 95% identical to a SEQ ID NO.:
- 1 or to a SEQ ID NO.:
- 3.
A method of expressing a green fluorescent protein in a host cell comprising the steps of:
- ligating one or more amplified nucleotides encoding the green fluorescent protein having at least 95% sequence homology to SEQ ID NOS.:
- 2, 4, 6, or 8, into a vector;
- inserting the vector into the host cell;
- and
- expressing the green fluorescent protein.
A method of isolating one or more expressed green fluorescent proteins comprising the steps of:
- plating the one or more expressed green fluorescent proteins having at least 95% sequence homology to SEQ ID NOS.:
- 2, 4, 6, or 8, onto a culture plate;
- incubating the culture plate at 37° C. for a period not less than 8 hours;
- selecting and picking one or more green fluorescent colonies from the culture plate;
- resuspending the selected green fluorescent colonies in water or a buffer;
- plating the resuspended green fluorescent colonies onto the culture plate;
- incubating the culture plate at room temperature for at least 48 hours;
- resuspending the green fluorescent colonies in the buffer;
- sonicating the green fluorescent colonies in the buffer on ice;
- centrifuging the sonicated buffer solution;
- separating the sonicated buffer solution to yield a cleared lysate and a cellular debris;
- and
- isolating the expressed green fluorescent protein using metal-affinity chromatography, chromatography, affinity extraction, or other protein purification techniques.
A kit comprising one or more containers, wherein at least one of the containers comprises an isolated nucleic comprising a nucleotide sequence that is at least 95% homologous to a SEQ ID NO.:
- 2 or to a SEQ ID NO.:
- 4, an isolated protein molecule comprising an amino acid sequence, wherein the amino acid sequence is at least 95% identical to a SEQ ID NO.:
- 1 or to a SEQ ID NO.:
- 3, or both.
Dependencies are inferred from the claim text.