US006410241B1 (12) United States Patent (10) Patent No.: US 6,410,241 B1 Sykes et al. (45) Date of Patent: Jun. 25, 2002 (54) METHODS OF SCREENING OPEN READING Nisson et al., “Rapid and efficient cloning of Alu-PCR FRAMES TO DETERMINE WHETHER THEY products using uracil DNA glycosylase,” PCR Methods ENCODE POLYPEPTIDES WITH AN Appl., 1:120-123, 1991. ABILITY TO GENERATE AN IMMUNE Rashtchian et al., “Uracil DNA glycosylase-mediated clon RESPONSE ing of polymerase chain reaction-amplified DNA. Applica tion to genomic and cDNA cloning,” Anal. Biochem, (75) Inventors: Kathryn F. Sykes; Stephen Albert 206:91-97, 1992. Johnston, both of Dallas, TX (US) Switzer et al., “Rapid Screening of open reading frames by (73) Assignee: Board of Regents, The University of protein Synthesis with an in Vitro transcription and transla Texas System, Austin, TX (US) tion assay,” Biotechniques, 18:244-248, 1995. 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Li et al (1997) Gene Therapy 4:449–454.* (57) ABSTRACT Johnston et al (1997) Vaccine 15: 808–809.* Barry et al (1995) Nature 377. 632–635.* The present invention relates to linear expression elements Logel et al., “Synthesis of crna probes from pcr-generated (LEES) and circular expression elements (CEES), which are dna,” Biotechniques, 13:604–610, 1992. useful in a variety of molecular biology protocols. Cassata et al., “Rapid expression Screening of Caenorhab Specifically, the invention relates to the use of LEEs and ditis elegans homeobox open reading frames using a CEES to Screen for gene function, biological effects of gene two-step polymerase chain reaction promoter-gfp reporter function, antigens, and promoter function. The invention contstruction technique,” Gene, 212:127-135, 1998. also provides methods of producing proteins, antibodies, Johnston and Barry, “Genetic to genomic vaccination,” antigens, and vaccines. Also, the invention relates to meth Vaccine, 15:808–809, 1997. ods of making LEEs and CEES, and LEEs and CEEs Kain et al., “Universal promoter for gene expression without produced by Such methods. cloning: Expression-PCR,” Biotechniques, 10:366-368, 370, 371, 372, 374, 1991. 28 Claims, 8 Drawing Sheets US 6,410,241 B1 Page 2 OTHER PUBLICATIONS Schickel et al., “Kluyveromyces lactis killer system: analysis Haun and MoSS, "Ligation-independent cloning of of cytoplasmic promoters of the linear plasmids, Nucleic. gluthathione S-transferase fusion genes for expression in Acids Res., 24:1879–1886, 1996. Escherichia coli,' Gene, 112:37-43, 1992. Schrinder and Meinhardt, “An extranuclear expression Sys Haun et al., “Rapid, reliable ligation-independent cloning of tem for analysis of cytoplasmic promoters of yeast linear pcr products using modified plasmid vectors, Biotech killer plasmids," Plasmid, 33:139-151, 1995. niques, 13:515-518, 1992. Schinder et al., “Extranuclear expression of the bacterial Kaluz and Flint, “Ligation-independent cloning of PCR Xylose isomerase (XylA) and the UDP-glucose deydroge products with primers containing nonbase residues,” Nucl. nase (hase) genes in yeast with Kluyveromyces lactis linear Acids Res., 22:4845, 1994. killer plasmids as vectors,” Curr. Microbiol., 33:323-330, Kamper et al., “Heterologous gene expression on the linear 1996. DNA killer plasmid from Kluyveromyces lactis,” Curr: Genet., 19:109-118, 1991. Sekine et al., “Identification and characterization of the La Flamme et al., “TrypanoSoma cruzi: expression of inter linear IS3 molecules generated by Staggered breaks,” J. Biol. leukin-2 utilizing both Supercoiled plasmids and linear Chem., 271:197-202, 1996. DNA’s.” Exp. Parasitol., 83:159–163, 1996. Sykes and Johnston, “Linear expression elements: a rapid, in Lobocka et al., “Characterization of the primary immunity Vivo, method to Screen for gene functions,” Nat. Biotech., region of the Escherichia coli linear plasmid prophage 17:355-359, 1999. N15, J. Bacteriol., 178:2902-2910, 1996. Tang et al., “Genetic immunization is a simple method for Mead et al., “A universal method for the direct cloning of eliciting an immune response,” Nature, 356:152-154, 1992, PCR amplified nucleic acid,” Biotechnology, 9:657-663, only pages 152 and 153 provided. 1991. Meinhardt et al., “A novel approach to express a heterolo Tanguy-Rougeau et al., “Expression of a foreign Km' gene gous gene Kluyveromyces lactis linear killer plasmids: in linear killer DNA plasmids in yeast,” Gene., 91:43-50, expression of the bacterial APH gene from a cytoplasmic 1990. promoter fragment without in-phase fusion to the plasmid Ton-Hoang et al., “Efficient transportation of IS911 circles open reading frame,” Plasmid., 32:318-327, 1994. in vitro, Embo. J., 17:1169–1181, 1998. Monoco et al., “Expression of recombinant human granu locyte colony-stimulating factor in CHO dhfr-cells: new Turner and Moyer, “A PCR-based method for manipulation insights into the in vitro amplification expression system.” of the Vaccinia virus genome that eliminates the need for Gene, 180: 145-150, 1996. cloning,” Bio Techniques, 13:764-771, 1992. Rashtchian, "Novel methods for cloning and engineering Xie and Tsong, “Study of mechanisms of electric field-in genes using the polymerase chain reaction, Curr. Opin. duced DNA transfection V. effects of DNA topology on Biotech., 6:30-36, 1995. Surface binding, cell uptake, expression, and integration into Sampath et al., “Versatile vectors for direct cloning and host chromosomes of DNA in the mammalian cell,” BioS ligation-independent cloning of PCR-amplified fragments phys. Jour., 65:1684–1689, 1993. for Surface displays on filamentous bacteriophages, Gene, 190:5-10, 1997. * cited by examiner U.S. Patent Jun. 25, 2002 Sheet 1 of 8 US 6,410,241 B1 bel Ou s MWOd O r S S s s S 901 x suaunt SS U.S. Patent Jun. 25, 2002 Sheet 2 of 8 US 6,410,241 B1 U.S. Patent Jun. 25, 2002 Sheet 3 of 8 US 6,410,241 B1 3OT Sll l/WO &D ?p OU S : d l CY 59G? Oll sC S d bel Ou >< s OOT-IT-IG Q &B l OOTAMO d SSO O wall s s - s s g0l. X sugun1 SS U.S. Patent Jun. 25, 2002 Sheet 4 of 8 US 6,410,241 B1 O r s s s g0l X suaunt 5 U.S. Patent Jun. 25, 2002 Sheet 5 of 8 US 6,410,241 B1 | 0000|| 000||~ 001§ SÈ •) $3 §S. > ~ 0||È U.S. Patent Jun. 25, 2002 Sheet 6 of 8 US 6,410,241 B1 s IAWOd C s S s 601 X suaunt U.S. Patent Jun. 25, 2002 Sheet 8 of 8 US 6,410,241 B1 S S S CS S s S U s CM) Q S. S. S CO CO s CN O apes/7 fu/suallInT Sol US 6,410,241 B1 1 2 METHODS OF SCREENING OPEN READING circular nucleic acid Segment comprising a promoter or FRAMES TO DETERMINE WHETHER THEY putative promoter and an ORF or putative ORF. These ENCODE POLYPEPTDES WITH AN methods may comprise: a) obtaining at least one linear or ABILITY TO GENERATE AN IMMUNE circular nucleic acid Segment comprising a promoter or RESPONSE putative promoter and an ORF encoding a peptide or puta tive ORF; b) placing the nucleic acid segment under con ditions conducive to expression of the polypeptide from the This application claims the benefit of U.S.