Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Utility Patent
1998-09-30
2001-01-02
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S091100, C435S091400, C435S091410, C435S252300, C435S183000, C435S320100, C435S325000, C536S023100, C536S023200, C536S023400
Utility Patent
active
06168919
ABSTRACT:
This invention relates to the field of preparing and screening libraries of clones containing microbially derived DNA and to protein, e.g. enzyme libraries and kits produced therefrom. More particularly, the present invention is directed to recombinant enzyme expression libraries, recombinant enzyme libraries and kits prepared therefrom which recombinant enzymes are generated from DNA obtained from microorganisms.
Industry has recognized the need for new enzymes for a wide variety of industrial applications. As a result, a variety of microorganisms have been screened to ascertain whether or not such microorganisms have a desired enzyme activity. If such microorganism does have a desired enzyme activity, the enzyme is then recovered from the microorganism.
Naturally occurring assemblages of microorganisms often encompass a bewildering array of physiological and metabolic diversity. In fact, it has been estimated that to date less than one percent of the world's organisms have been cultured. It has been suggested that a large fraction of this diversity thus far has been unrecognized due to difficulties in enriching and isolating microorganisms in pure culture. Therefore, it has been difficult or impossible to identify or isolate valuable enzymes from these samples. These limitations suggest the need for alternative approaches to characterize the physiological and metabolic potential i.e. activities of interest of as-vet uncultivated microorganisms, which to date have been characterized solely by analyses of PCR amplified rRNA Rene fragments, clonally recovered from mixed assemblage nucleic acids.
In accordance with one aspect of the present invention, there is provided a novel approach for obtaining enzymes for further use, for example, for packaging into kits for further research. In accordance with the present invention, recombinant enzymes are generated from microorganisms and are classified by various enzyme characteristics. In this manner, the enzymes can be provided as packaged enzyme screening kits, with enzymes in the kit being grouped to have selected enzyme characteristics.
More particularly, in accordance with this aspect of the present invention there is provided a recombinant expression library which is comprised of a multiplicity of clones which are capable of expressing recombinant enzymes. The expression library is produced by recovering DNA from a microorganism, cloning such DNA into an appropriate expression vector which is then used to transfect or transform an appropriate host for expression of a recombinant protein.
Thus, for example, genomic DNA may be recovered from either a culturable or non-culturable organism and employed to produce an appropriate recombinant expression library for subsequent determination of enzyme activity.
In accordance with an aspect of the present invention, such recombinant expression library may be prepared without prescreening the organism from which the library is prepared for enzyme activity.
Having prepared a multiplicity of recombinant expression clones from DNA isolated from an organism, the polypeptides expressed by such clones are screened for enzyme activity and specified enzyme characteristics in order to identify and classify the recombinant clones which produce polypeptides having specified enzyme characteristics.
In one aspect, the invention provides a process of screening clones having DNA from an uncultivated microorganism for a specified protein, e.g. enzyme, activity which process comprises:
screenings for a specified protein, e.g. enzyme, activity in a library of clones prepared by
(i) recovering DNA from a DNA population derived from at least one uncultivated microorganism, and
(ii) transforming a host with recovered DNA to produce a library of clones which are screened for the specified protein, e.g. enzyme, activity.
The library is produced from DNA which is recovered without culturing of an organism, particularly where the DNA is recovered from an environmental sample containing microorcanisms which are not or cannot be cultured.
In a preferred embodiment of this aspect DNA is ligated into a vector. particularly wherein the vector further comprises expression regulatory sequences which can control and regulate the production of a detectable enzyme activity from the ligated DNA.
The f-factor (or fertility factor) in
E. coli
is a plasmid which effects high frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself. To archive and stably propogate large DNA fragments from mixed microbial samples, a particularly preferred embodiment is to use a cloning vector containing an f-factor origin of replication to generate genomic libraries that can be replicated with a high degree of fidelity. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable “environmental DNA library.”
In another preferred embodiment, double stranded DNA obtained from the uncultivated DNA population is selected by:
converting the double stranded genomic DNA into single stranded DNA;
recovering from the converted single stranded DNA single stranded DNA which specifically binds, such as by hybridization, to a probe DNA sequence; and
converting recovered single stranded DNA to double stranded DNA.
The probe may be directly or indirectly bound to a solid phase by which it is separated from single stranded DNA which is not hybridized or otherwise specifically bound to the probe.
The process can also include releasing single stranded DNA from said probe after recovering said hybridized or otherwise bound single stranded DNA and amplifying the single stranded DNA so released prior to converting it to double stranded DNA.
The invention also provides a process of screening clones having DNA from an uncultivated microorganisms for a specified protein, e.g. enzyme, activity which comprises screening for a specified gene cluster protein product activity in the library of clones prepared by: (i) recovering DNA from a DNA population derived from at least one uncultivated microorganism; and (ii) transforming a host with recovered DNA to produce a library of clones with the screens for the specified protein, e.g. enzyme, activity. The library is produced from gene cluster DNA which is recovered without culturing of an organism, particularly where the DNA gene clusters are recovered from an environmental sample containing microorganisms which are not or cannot be cultured.
Alternatively, double-stranded gene cluster DNA obtained from the uncultivated DNA population is selected by converting the double-stranded genomic gene cluster DNA into single-stranded DNA; recovering from the converted single-stranded gene cluster polycistron DNA, single-stranded DNA which specifically binds, such as by hybridization, to a polynucleotide probe sequence; and converting recovered single-stranded gene cluster DNA to double-stranded DNA.
These and other aspects of the present invention are described with respect to particular preferred embodiments and will be apparent to those skilled in the art from the teachings herein.
REFERENCES:
patent: 5171684 (1992-12-01), Yen et al.
patent: 5712146 (1998-01-01), Khosla et al.
patent: 5958672 (1999-09-01), Short
Lactic Dehydrogenase, Sigma catalog, p. 634, 1997.
Anderson et al., Met. Enzymol., vol. 68, pp. 428-436, 1979.
Promega Catalog, p. 205, 1993.
Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, vol. 2, p. 9.30 and pp. 12.1-12.20, 1989.*
Achutamurthy Ponnathapu
Diversa Corporation
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Tung Peter P.
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