Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-03-03
2002-04-02
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S455000, C435S463000, C436S094000
Reexamination Certificate
active
06365377
ABSTRACT:
COPYRIGHT NOTIFICATION
Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
The present invention relates to molecular shuffling, and to splicing of nucleic acids and proteins.
BACKGROUND OF THE INVENTION
Nucleic acid shuffling provides for the rapid evolution of nucleic acids, in vitro and in vivo. Rapid evolution provides for the commercial production of encoded molecules (e.g., nucleic acids and proteins) with new and/or improved properties. Proteins and nucleic acids of industrial, agricultural and therapeutic value can be created or improved through shuffling procedures. A number of publications by the inventors and their co-workers describe nucleic acid shuffling and applications of this technology. For example, Stemmer et al. (1994) “Rapid Evolution of a Protein”
Nature
370:389-391; Stemmer (1994) “DNA Shuffling by Random Fragmentation and Reassembly: in vitro Recombination for Molecular Evolution,”
Proc. Natl. Acad. USA
91:10747-10751; Stemmer U.S. Pat. No. 5,603,793 METHODS FOR IN VITRO RECOMBINATION; Stemmer et al. U.S. Pat. No. 5,830,721 DNA MUTAGENESIS BY RANDOM FRAGMENTATION AND REASSEMBLY; Stemmer et al., U.S. Pat. No. 5,811,238 METHODS FOR GENERATING POLYNUCLEOTIDES HAVING DESIRED CHARACTERISTICS BY ITERATIVE SELECTION AND RECOMBINATION describe, e.g., in vivo and in vitro nucleic acid, DNA and protein shuffling in a variety of formats, e.g., by repeated cycles of mutagenesis, shuffling and selection, as well as methods of generating libraries of displayed peptides and antibodies.
Applications of DNA shuffling technology have also been developed by the inventors and their co-workers. In addition to the publications noted above, Minshull et al., U.S. Pat. No. 5,837,458 METHODS AND COMPOSITIONS FOR CELLULAR AND METABOLIC ENGINEERING provides for the evolution of metabolic pathways and the enhancement of bioprocessing through recursive shuffling techniques. Crameri et al. (1996), “Construction And Evolution Of Antibody-Phage Libraries By DNA Shuffling”
Nature Medicine
2(1):100-103 describe, e.g., antibody shuffling for antibody phage libraries. Additional details regarding DNA Shuffling can also be found in WO95/22625, WO97/20078, WO96/33207, WO97/33957, WO98/27230, WO97/35966, W098/ 31837, WO98/13487, WO98/13485 and WO989/42832.
Physical nucleic acid shuffling techniques (as opposed, e.g., to “in silico” methods which are performed, at least in part, by manipulation of character strings in a computer) rely upon actual recombination between physical nucleic acids, whether the format is an in vitro or an in vivo format. Recombination occurs at a relatively high frequency, e.g., where there are complementary nucleic acids between strands to be recombined. Thus. nucleic acids to be recombined are typically e.g., about 70% identical/complementary in sequence over regions of, e.g., about 30-40 nucleotides. It would be desirable to be able to recombine low homology, or even non-homologous sequences, thereby increasing access to the potential sequence space encoded by recombinant nucleic acids resulting from shuffling methods. For example, for proteins which are commercially valuable, it would be desirable to be able to gain access to a recombination/mutation spectrum which is different than that of the native protein to provide for greater diversity in products produced by the various available shuffling strategies.
Similarly, nucleic acid recombination generally can be difficult to modulate, resulting in regions of high or low crossover frequency between two different targets for recombination. The crossover frequency for a particular pairing of sequences on two different targets is one feature that mediates the recombinant nucleic acids that result from recombination methods. Improved methods of modulating the recombination frequency at potential recombination sites would be desirable to weight/bias recombination product outcomes.
In general, new techniques which facilitate, improve or add levels of control to recombination methods are highly desirable. In particular, techniques which permit shuffling of divergent nucleic acids, or which provide for modulation and tuning of shuffling rates are desirable. The present invention provides such significant new recombination protocols, as well as other features which will be apparent upon complete review of this disclosure.
SUMMARY OF THE INVENTION
The present invention provides a number of new nucleic acid recombination formats for nucleic acid shuffling. In the methods, a number of insertion sequences are inserted into one or more parental nucleic acid to provide a modified target nucleic acid substrate for recombination and subsequent mutation. The number, type and placement of such insertion sequences provides for the ability to shuffle nucleic acids with little or no homology other than the insertion sequences. In addition, these insertion sequences provide for the ability to modulate or “tune” recombination frequencies between target nucleic acids. The methods typically take advantage of self-splicing, trans-splicing or use cellular machinery to remove the insertion sequences from final coded nucleic acids or proteins, e.g., where the insertion sequences are introns, inteins, proteolyzed polypeptide sequences or the like. The insertion sequences can also comprise markers, molecular tags, or the like, e.g., for purification of encoded molecules or can serve to allow for expression of otherwise toxic proteins (e.g., RNases, Dnases, restriction enzymes, proteases, lipases, recombinases, ligases, polymerases, etc.) e.g., in a form where an intein is excised in vivo. Similarly, in vitro expression of insertion modified sequences can result in the production of these and other proteins in vitro, e.g., using in vitro expression systems.
Methods of shuffling two target nucleic acids (i.e., a first and a second target nucleic acid) are provided. In the methods, a first and a second target nucleic acid are provided, e.g., by cloning, PCR amplification, synthesis, isolation from an environmental source (soil, air, water, etc.), or other methods. At least one of the first and second target nucleic acids (and typically both) have a plurality of homologous or non-homologous insertion nucleic acid sequences, such as one or more intron (e.g., self-splicing bacterial, eukaryotic or trans-splicing intron), intein, subsequence removed by site specific recombination (e.g., similar to V-D-J recombination for antibody production), or the like, optionally including intron splicing enhancers or the like. The target nucleic acids are recombined, producing a shuffled recombinant nucleic acid.
In addition to providing for new recombination methods per se, the invention also provides methods of producing selected proteins and RNAs, for any of the purposes that such proteins and RNAs are ordinarily produced. For example, in one aspect, a first shuffled nucleic acid subsequence encoding a first portion of the selected protein and a second nucleic acid subsequence encoding a second portion of the selected protein is provided. The nucleic adds can be on the same strand (as in cis-mediated reactions) or on different strands (as in trans mediated reactions). The first and second subsequences are expressed to produce a first protein subsequence and a second protein subsequence, which are spliced to produce the selected protein. Commonly, more than two subsequences are spliced, e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more sequences, as set forth herein. The splicing reaction can be in cis or in trans (or both) and can be in viro or in vivo (or both). Splicing can occur by spontaneous or controlled mechanisms.
Similarly, in RNA production methods, a first shuffled nucleic acid subsequence encoding a
Heinrichs Volker
Patten Phillip A.
Stemmer Willem P. C.
Kruse Norman J.
Law Offices of Jonathan Alan Quine
Lu Frank
Maxygen Inc.
Quine Jonathan Alan
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