Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-08-17
2004-09-14
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S014000, C435S015000, C435S016000, C435S018000, C435S019000, C435S021000, C435S022000, C435S023000, C435S024000
Reexamination Certificate
active
06790605
ABSTRACT:
This invention relates to the field of protein engineering. More particularly, the invention relates to the directed mutagenesis of DNA and screening of clones containing the mutagenized DNA for resultant specified protein, particularly enzyme, activity(ies) of interest.
In one aspect the invention provides a process for obtaining an enzyme having a specified enzyme activity derived from a heterogeneous DNA population, which process comprises: screening, for the specified enzyme activity, a library of clones containing DNA from the heterogeneous DNA population which have been exposed to directed mutagenesis towards production of the specified enzyme activity.
Another aspect of the invention provides a process for obtaining an enzyme having a specified enzyme activity, which process comprises: screening, for the specified enzyme activity, a library of clones containing DNA from a pool of DNA populations which have been exposed to directed mutagenesis in an attempt to produce in the library of clones DNA encoding an enzyme having one or more desired characteristics, which can be the same or different from the specified enzyme activity. In a preferred embodiment, the DNA pool which is subjected to directed mutagenesis is a pool of DNA which has been selected to encode enzymes having at least one enzyme characteristic, in particular at least one common enzyme activity.
Also provided is a process for obtaining a protein having a specified activity derived from a heterogeneous population of gene clusters by screening, for the specified protein activity, a library of clones containing gene clusters from the heterogeneous gene cluster population which have been exposed to directed mutagenesis towards production of specified protein activities of interest.
Also provided is a process of obtaining a gene cluster protein product having a specified activity, by screening, for the specified protein activity, a library of clones containing gene clusters from a pool of gene cluster populations which have been exposed to direct mutagenesis to produce in the library of clones gene clusters encoding proteins having one or more desired characteristics, which can be the same or different from the specified protein activity. Preferably, the pool of gene clusters which is subjected to directed mutagenesis is one which has been selected to encode proteins having enzymatic activity in the synthesis of at least one therapeutic, prophylactic or physiological regulatory activity.
The process of either of these aspects can further comprise, prior to the directed mutagenesis, selectively recovering from the heterogeneous population of gene clusters, gene clusters which comprise polycistronic sequences coding for proteins having at least one common physical, chemical or functional characteristic which can be the same or different from the activity observed prior to directed mutagenesis. Preferably, recovering the gene cluster preparation comprises contacting the gene cluster population with a specific binding partner, such as a solid phase-bound hybridization probe, for at least a portion of the gene cluster of interest. The common characteristic of the resultant protein(s) can be classes of the types of activity specified above, i.e., such as a series of enzymes related as parts of a common synthesis pathway or proteins capable of hormonal, signal transduction or inhibition of metabolic pathways or their functions in pathogens and the like. The gene cluster DNA is recovered from clones containing such gene cluster DNA from the heterogeneous gene cluster population which exhibit the activity of interest. Preferably, the directed mutagenesis is site-specific directed mutagenesis. This process can further include a step of pre-screening the library of clones for an activity, which can be the same or different from the specified activity of interest, prior to exposing them to directed mutagenesis. This activity can result, for example, from the expression of a protein or related family of proteins of interest.
The process of any of these aspects can further comprise, prior to said directed mutagenesis, selectively recovering from the heterogeneous DNA population DNA which comprises DNA sequences coding for enzymes having at least one common characteristic, which can be the same or different from the specified enzyme activity. Preferably, recovering the DNA preparation comprises contacting the DNA population with a specific binding partner, such as a solid phase bound hybridization probe, for at least a portion of the coding sequences. The common characteristic can be, for example, a class of enzyme activity, such as hydrolase activity. DNA is recovered from clones containing DNA from the heterogeneous DNA population which exhibit the class of enzyme activity. Preferably, the directed mutagenesis is site-specific directed mutagenesis. The process of this aspect can further include a step of prescreening the library of clones for an activity, which can be the same or different from the specified enzyme activity, prior to exposing them to directed mutagenesis. This activity can result, for example, from the expression of a protein of interest.
The heterogeneous DNA population from which the DNA library is derived is a complex mixture of DNA, such as is obtained, for example, from an environmental sample. Such samples can contain unculturable or uncultured multiple or single organisms. These environmental samples can be obtained from, for example, Arctic and Antarctic ice, water or permafrost sources, materials of volcanic origin, materials from soil or plant sources in tropical areas, etc. A variety of known techniques can be applied to enrich the environmental sample for organisms of interest, including differential culturing, sedimentation gradient, affinity matrices, capillary electrophoresis, optical tweezers and fluorescence activated cell sorting. The samples can also be cultures of a single organism.
The microorganisms from which the libraries may be prepared include prokaryotic microorganisms, such as Eubacteria and Archaebacteria, and lower eukaryotic microorganisms such as fungi, some algae and protozoa. The microorganisms are uncultured microorganisms obtained from environmental samples and such microorganisms may be extremophiles, such as thermophiles, hyperthermophiles, psychrophiles, psychrotrophs, etc.
Bacteria and many eukaryotes have a coordinated mechanism for regulating genes whose products are involved in related processes. The genes are clustered, in structures referred to as “gene clusters,” on a single chromosome and are transcribed together under the control of a single regulatory sequence, including a single promoter which initiates transcription of the entire cluster. The gene cluster, the promoter, and additional sequences that function in regulation altogether are referred to as an “operon” and can include up to 20 or more genes, usually from 2 to 6 genes. Thus, a gene cluster is a group of adjacent genes that are either identical or related, usually as to their function.
Some gene families consist of identical members. Clustering is a prerequisite for maintaining identity between genes, although clustered genes are not necessarily identical. Gene clusters range from extremes where a duplication is generated to adjacent related genes to cases where hundreds of identical genes lie in a tandem array. Sometimes no significance is discernable in a repetition of a particular gene. A principal example of this is the expressed duplicate insulin genes in some species, whereas a single insulin gene is adequate in other mammalian species.
It is important to further research gene clusters and the extent to which the full length of the cluster is necessary for the expression of the proteins resulting therefrom. Further, gene clusters undergo continual reorganization and, thus, the ability to create heterogeneous libraries of gene clusters from, for example, bacterial or other prokaryote sources is valuable in determining sources of novel proteins, particularly including enzymes such as, for examp
Diversa Corporation
Nashed Nashaat T.
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