Method of synthesizing a fluorescently labeled protein

Details Method of synthesizing a fluorescently labeled protein Method of synthesizing a fluorescently labeled protein

1996-01-24

2002-09-10

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

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

C435S068100, C435S006120

Reexamination Certificate

active

06448033

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology, biotechnology and protein synthesis. More specifically, the present invention relates to a novel method of synthesizing a fluorescently labeled protein in a cell-free protein synthesis system.
2. Description of the Related Art
Several lines of evidence indicate that nascent polypeptides acquire secondary and tertiary structure on ribosomes. Beckmann and his coworkers (1990) provided evidence that Hsp70 interacts with nascent proteins and postulated cotranslational folding. The ribosome itself may play an important role in the folding process. Lim and Spirin (1986) hypothesized that a nascent peptide must be synthesized as an &agr; helix. The use of fluorescence techniques to study extension and folding of nascent peptides and proteins on ribosomes has been reviewed. Previously, these techniques were applied to demonstrate nascent MS2 protein folds while bound to ribosomes. Chaperones have been shown to facilitate folding of many proteins from their denatured state, but very little is known of whether these proteins function on ribosomes during translation. Hartl and coworkers presented data indicating that DnaJ is the first chaperone that binds to the nascent peptides of firefly luciferase or chloramphenicol acetyl transferase on wheat germ ribosomes (Hendrick et al., 1993).
The prior art is deficient in the lack of effective means synthesizing a fluorescently labeled protein in a cell-free protein synthesis system. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
In the present invention, coumarin from coumarin-maleimidyl-SAcMet-tRNA
f
(CPM-SAcMet-tRNA
f
) was incorporated into the N-terminus of nascent polypeptides that were synthesized on ribosomes in a bacterial cell-free coupled transcription/translation system. Fluorescence techniques were used to monitor changes in the local environment and mobility of the N-terminal probe and in turn effects of the chaperones on folding, activation and release of the nascent or full-length polypeptides from the ribosomes. The present invention shows that the chaperones affect folding of nascent rhodanese polypeptides while these are bound as peptidyl-tRNA on ribosomes. The chaperones facilitate their release from the ribosomes as enzymatically active protein.
In one embodiment of the present invention, there is provided a method of synthesizing a fluorescently labeled protein in a cell-free protein synthesis system, comprising the steps of: (a) incubating a sample of ribosomes obtained from a cell-free extract with plasmid DNA containing a coding sequence for a protein of interest, said sample incubated in a coupled transcription/translation medium together with an aminoacyl tRNA having fluorescent label; (b) partially purifying said fluorescently labeled protein by separating newly synthesized, fluorescently-labeled protein from other fluorescent components within said sample; (c) measuring the amount of protein synthesized; (d) determining fluorescence of newly synthesized protein and (e) determining the biological activity of the newly synthesized protein.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.


REFERENCES:
patent: 5258862 (1993-11-01), Hardesty et al.
Kudlicki, W. et al. (1992) “High efficiency cell-free synthesis of proteins: Refinement of the coupled transcription/translation system”Anal. Biochem. 206:389-393.*
Picking, W.D. et al. (1992) “Fluorescence characterization of the environment encountered by nascent polyalanine and polyserine as they exitEscherichia coliribosomes during translation”Biochem. 31:2368-2375.*
Hildenbrand, K. et al. (1982) “Sugar transport by the bacterial phosphotransferase system”J. Biol. Chem. 257(23):14518-14525.*
Stryer, L. (1988)Biochemistry. W.H. Freeman and Company, New York. pp. 752-758.

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