Lambdoid bacteriophage vectors for expression of foreign proteins

#The present invention relates to cloning vectors and methods for producing a library of DNA molecules capable of expressing a fusion polypeptide on the surface of a lambdoid phage particle.

#The expression of polypeptides fused to the surface of filamentous bacteriophage provides a powerful method for recovering a particular sequence from a large ensemble of clones (Smith et al., Science, 228:1315-1517, 1985). Peptides binding to avidin or antibodies have been selected from large libraries by the relatively simple method of panning (Scott et al., Science, 249:386-290, 1990; Devlin et al., Science, 249:404-406, 1990; and Cwirla et al., Proc. Natl. Acad. Sci. U.S.A., 87:6378-6382, 1990). Larger proteins, such as antibodies (McCafferty et al., Nature, 348:552-554, 1990; Lowman et al., Biochemistry, 30:10832-10838, 1992; and Kang et al., Proc. Natl. Acad. Sci. U.S.A., 88:4363-4366, 1991) and human growth hormone (Bass et al., Proteins, 8:309-314, 1990), have also been fused to surface proteins of filamentous phage which can then be used for the selection of particular sequences from a large number of variants.

#In these filamentous phage systems, the foreign protein or polypeptide is fused to the amino-terminus of either coat protein III or coat protein VIII of the filamentous phage M13 and the fused protein is secreted through the Escherichia coli cytoplasmic membrane into the periplasmic space. Most of the proteins successfully fused with filamentous phage surface proteins have been secreted proteins and, although Rebar and Pabo (Science, 263:671-673, 1994) have recently used pIII to display three zinc fingers of Zif268 (a DNA-binding protein), many cytoplasmic proteins will interfere with the passage of the fusion product from the cytoplasm to the periplasm.

#In the drawings forming a portion of this disclosure:

#FIG. 1 illustrates a flow chart of the construction of the λV′ sac vector. Also indicated is the partial nucleotide and amino acid sequence of the λV′ sac vector. The amino acid sequence is given in SEQ ID NO 9. The top strand of the nucleotide sequence from left to right is given in SEQ ID NO 10. The bottom strand of the nucleotide sequence from left to right is given in SEQ ID NO 11. The left arm of λ phage is depicted with genes Nu1 through J shown as boxes. The arrow at the top of the figure indicates the number of kilobase pairs (kb) of the λ vector. The DNA segment from nucleotide 5505 to 10,325 and encoding genes Nu3 through G was removed from the λ vector by digestion with the enzymes BamHI and NsiI. This fragment is shown as a line with nucleotide numbers below the topmost λ genetic map. Insertion of the DNA fragment into the M13 mp19 and pUC12 vectors, site-directed mutagenesis to introduce the amber stop codon and Sac I restriction site, and subsequent combination with the λ2000 vector to create the λV′ sac vector are as described in Example 1a1. A portion of the resulting λV′ sac vector is shown at the bottom of the figure consisting of the amino acid and nucleotide sequence of part of the mutagenized V gene. The nucleotide positions which were mutagenized are underlined and a newly created Sac I site is indicated in boldface type. The amber suppressor stop codon is indicated by the nucleotide sequence TAG which is underlined. Numbers at the left side from top to bottom indicate the amino acid number of the pV gene and the nucleotide sequence from the left arm of λ, respectively. Abbreviations used to indicate the corresponding restriction sites are: Ba, BamHI; Bc, BclI; Aa, AatII; Ap, ApaI; N, NsiI; E, Ecori: and ApL, ApaLI.

#FIG. 2 illustrates partial sequence of the λblue and λfoo vectors. The partial sequences illustrate the amino acid and nucleotide sequence between the amber stop codon of the pV protein, indicated by TAG and underlined, and the downstream restriction sites. The names of the restriction sites are indicated below the sites and the nucleotide sequences defining the restriction sites are underlined. The corresponding amino acid sequence is given above the nucleotide sequence for each of λV′mcs, λblue and λfoo. The λV′mcs amino acid and nucleotide sequences are given as SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The λblue amino acid and nucleotide sequences are given as SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The λfoo amino acid and nucleotide sequences are given from amino acid residues 178 to 237 of SEQ ID NO 6 and base number 547 to 726 of SEQ ID NO 5, respectively. The amino acid sequence downstream of the suppressor codon is expressed at the carboxy terminus of pV in the presence of a suppressor tRNA as described in Example 1a2. Numbers in the right-hand column, from top to bottom, indicate the amino acid position from the start codon of the pV gene and the nucleotide sequence of the λ vector, from the beginning of the left arm, at the end of the respective lines.

#Amino Acid Residue: An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are preferably in the “L” isomeric form. However, residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature (described in J. Biol. Chem., 243:3552-59 (1969) and adopted at 37 CFR §1.822(b)(2)), abbreviations for amino acid residues are shown in the following Table of Correspondence:

#TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Ala alanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine V Val valine P Pro proline K Lys lysine H His histidine Q Gln glutamine E Glu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine D Asp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine X Xaa Unknown or other

#It should be noted that all amino acid residue sequences represented herein by formulae have a left-to-right orientation in the conventional direction of amino terminus to carboxy terminus. In addition, the phrase “amino acid residue” is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those listed in 37 CFR 1.822(b)(4), and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to an amino-terminal group such as NH2 or acetyl or to a carboxy-terminal group such as COOH.