Phagemid vectors

Abstract

phagemid vectors containing a sequence of features between a Col E1 origin and an f1 origin are useful for display of polypeptides or proteins, including antibody libraries.

Description

This application claims priority under 35 USC 119(e) to provisional application 60/287,355, filed Apr. 27, 2001.

BACKGROUND

1. Technical Field

This disclosure relates to cloning vectors. More specifically, phagemid vectors useful in the cloning and expression of foreign genetic information are disclosed.

2. Background of Related Art

Plasmids are extrachromosomal genetic elements capable of autonomous replication within their hosts. Bacterial plasmids range in size from 1 Kb to 200 Kb or more and encode a variety of useful properties. Plasmid encoded traits include resistance to antibiotics, production of antibiotics, degradation of complex organic molecules, production of bacteriocins, such as colicins, production of enterotoxins, and production of DNA restriction and modification enzymes.

Although plasmids have been studied for a number of years in their own right, particularly in terms of their replication, transmissibility, structure and evolution, with the advent of genetic engineering technology the focus of plasmid research has turned to the use of plasmids as vectors for the cloning and expression of foreign genetic information. In its application as a vector, the plasmid should possess one or more of the following properties. The plasmid DNA should be relatively small but capable of having relatively large amounts of foreign DNA incorporated into it. The size of the DNA insert is of concern in vectors based on bacteriophages where packing the nucleic acid into the phage particles can determine an upper limit. The plasmid should be under relaxed replication control. That is, where the replication of the plasmid molecule is not strictly coupled to the replication of the host DNA (stringent control), thereby resulting in multiple copies of plasmid DNA per host cell. The plasmid should express one or more selectable markers, such as the drug resistance markers, mentioned above, to permit the identification of host cells which contain the plasmid and also to provide a positive selection pressure for the maintenance of the plasmid in the host cell. Finally the plasmid should contain a single restriction site for one or more endonucleases in a region of plasmid which is not essential for plasmid replication. A vector as described above is useful, for example, for cloning genetic information, by which is meant integrating a segment of foreign DNA into the vector and reproducing identical copies of that information by virtue of the replication of the plasmid DNA.

The next step in the evolution of vector technology was the construction of so-called expression vectors. These vectors are characterized by their ability not only to replicate the inserted foreign genetic information but also to promote the transcription of the genetic information into mRNA and its subsequent translation into protein. This expression requires a variety of regulatory genetic sequences including but not necessarily limited to promoters, operators, transcription terminators, ribosomal binding sites and protein synthesis initiation and termination codons. These expression elements can be provided with the foreign DNA segment as parts thereof or can be integrated within the vector in a region adjacent to a restriction site so that when a foreign DNA segment is introduced into the vector it falls under the control of those elements to which it is now chemically joined.

Filamentous bacteriophage consist of a circular, single-stranded DNA molecule surrounded by a cylinder of coat proteins. There are about 2,700 molecules of the major coat proteins pVIII that envelope the phage. At one end of the phage particle, there are approximately five copies of each of gene III and VI proteins (pIII and pVI) that are involved in host cell binding and in the termination of the assembly process. The other end contains five copies of each of pVII and pIX that are required for the initiation of assembly and for maintenance of virion stability. In recent years, vectors have been developed and utilized for the display of foreign peptides and proteins on the surface of filamentous phage or phagemid particles.

The display of peptides and proteins on the surface of phage or phagemid particles represents a powerful methodology for selection of rare members in a complex library and for carrying out molecular evolution in the laboratory. The ability to construct libraries of enormous molecular diversity and to select for molecules with predetermined properties has made this technology applicable to a wide range of problems. A few of the many applications of such technology are: i) phage display of natural peptides including, mapping epitopes of monoclonal and polyclonal antibodies and generating immunogens; ii) phage display of random peptides, including mapping epitopes of monoclonal and polyclonal antibodies, identifying peptide ligands, and mapping substrate sites for proteases and kinases; and iii) phage display of protein and protein domains, including directed evolution of proteins, isolation of antibodies and cDNA expression screening.

Vectors have been developed which incorporate DNA from plasmids and bacteriophage. These phagemid vectors are derived by modifications of a plasmid genome containing an origin of replication from a bacteriophage, (e.g. f1, M13, fd) as well as the plasmid origin of replication. Phagemids are useful for the expression of foreign genetic information.

One known phagemid vector is
and
5′ CCT GAA TTC AAT TGT TAT CCG CTC ACA ATT CCA C 3′ (SEQ. ID. NO. 2).

The 2424 bp fragment and the 209 bp fragment were combined in a three-way ligation reaction with two overlapping oligonucleotides which contain a Not I, EcoR I and Pvu I sites to form a first intermediate plasmid (designated p110-81.6). (See FIG. 3.) The oligonucleotides used for this reaction were:
5′ CGG TAA TGC GGC CGC TAC ATG 3′ (SEQ. ID. NO. 3);
and
5′ AAT TCA TGT AGC GGC CGC ATT ACC GAT 3′ (SEQ. ID. NO. 4).

The resulting plasmid p110-81.6 was digested and sequenced in the altered region to identify a clone with the correct incorporation of the lac promoter, Pvu I, Sap I, EcoR, and Not I sites. The sequencing of p110-81.6 revealed a nucleic acid change at position 875 within the lac promoter. The published sequence of pBS II KS+ had an adenine at position 875. However, sequencing of p110-81.6 and the original pBS II KS+ revealed a guanine at position 875. The sequence (Seq. ID No. 19) of intermediate plasmid p110-81.6 is shown in FIGS. 4A-C.

Insertion of Terminator

A transcription termination sequence was inserted into the first intermediate plasmid (p110-81.6) upstream of the lac promoter at the Sap I site. (See FIG. 5.)

Plasmid 110-81.6 was digested with Sap I to create an insertion point for the oligonucleotides which contained a t_HP terminator (Nohno et al., Molecular and General Genetics, Vol. 205, pages 260-269 (1986). The oligonucleotides used in this ligation were:
5′ AGC GTA CCC GAT AAA AGC GGC TTC CTG ACA GGA GGC CGT TTT GTT TTG CAG CCC ACC T 3′; (SEQ. ID. No. 5);
and
5′ GCT AGG TGG GCT GCA AAA CAA AAC GGC CTC CTG TCA GGA AGC CGC TTT TAT CGG GTA C 3′ (SEQ. ID. NO. 6).

The resulting intermediate vector (designated p131-03.7) was digested and sequenced in the altered region to determine its identity. The sequence (Seq. ID No. 20) of intermediate vector p131-03.7 is shown in FIGS. 6A-C.

Insertion of Multiple Restriction Sites

Oligonucleotides containing the Xba I, XhoI, SpeI and Sfi sites were then inserted into intermediate plasmid p131-03.7.(See FIG. 7.)

Intermediate vector p131-03.7 was digested with EcoR I and Not I and then gel purified. Then overlapping oligonucleotides containing the Xba I, Xho I, Spe I and Sfi I sites were ligated into the p131-03.7 backbone. The oligonucleotides inserted were:
5′ AAT TCA CAT CTA GAT ATC TCG AGT CAA TAC TAG TGG CCA GGC CGG CCA GC 3′ (SEQ. ID. NO. 7);
and
5′ GGC CGC TGG CCG GCC TGG CCA CTA GTA TTG ACT CGA GAT ATC TAG ATG TG 3′ (SEQ. ID. NO. 8).

The resulting intermediate plasmid (designated p131-39.1) was sequenced and analyzed to determine its identity. The sequence (Seq. ID No. 21) of intermediate plasmid p131-39.1 is shown in FIGS. 8A-C.

Construction of Nucleotide Sequence Encoding Display Protein

Single stranded DNA from phage f1 (ATCC #15766-B2) was used as a template for the cloning of gene III. (Sec FIG. 9.)

The primers used were:
5′ AGT GGC CAG GCC GGC CTT GAA ACT GTT GAA AGT TGT TTA GCA AA 3′ (SEQ. ID. NO. 9)
which contains the Sfi I site, bases to maintain the coding frame and a portion of gene III; and
5 TCT GCG GCC GCT TAG CTA GCT TAA GAC TCT TTA TTA CGC AGT ATG TTA GCA 3′ (SEQ. ID. NO. 10);
which contains the end of gene III in which an internal ribosome binding site ordinarily used for the next downstream gene has been removed by changing a silent third base position in the corresponding codon. This oligonucleotide also contains a stop codon, Nhe I site for potential use in removal of the fusion, a second stop codon for use with the fusion, and the Not I site for cloning. The PCR fragment was digested with Sfi I and Not I and inserted into p131-39.1 digested with Sfi I and Not I to create intermediate vector p131-44.2. The integrity of the gene III region and flanking sequences was confirmed by sequence analysis.
Creation of the Upstream Transcriptional Control Cassette

Plasmid 131-39.1 was utilized as a shuttle vector for cloning the oligonucleotides containing the ompA signal peptide coding sequence. The upstream transcriptional control cassette was generated within intermediate plasmid 131-39.1 by inserting a pair of oligonucleotides which contain EcoR I, the ompA signal peptide leader, followed by a Sac I site, a small stuffer region, and a ribosome binding site. (See FIG. 9.) The oligonucleotides used were:

Eco Xba:
5′ AAT TCA AGG AGT TAA TTA TGA AAA AAA CCG CGA TTG CGA TTG CGG TGG CGC TGG CGG GCT TTG CGA CCG TGG CCC AGG CGG CCG AGC TCA TCT T 3′ (SEQ. ID. NO. 11);
and
Xba Eco:
5′ CTA GAA GAT GAG CTC GGC CGC CTG GGC CAC GGT CGC AAA GCC CGC CAG CGC CAC CGC AAT CGC AAT CGC GGT TTT TTT CAT AAT TAA CTC CTT G 3′ (SEQ. ID. NO. 12).
The RBS and leader sequences included in the upstream transcriptional control cassette are optimized for use in E. coli. These novel sequences are:
5′ AAG GAG 3′ (Seq. ID No.13)
for the RBS; and
5′ ATG AAA AAA ACC GCG ATT GCG ATT GCG GTG GCG CTG GCG GGC TTT GCG ACC GTG GCC CAG GCG GCC 3′ (Seq. ID No. 14)
for the ompA leader. The resulting plasmid was sequenced to confirm the identity of the insert and digested at the EcoRI and Xbal sites to generate a 94 bp fragment which is the upstream transcriptional control cassette.
Creation of the Downstream Transcriptional Control Cassette

Intermediate plasmid 131-39.1 was utilized as a shuttle vector for cloning the oligonucleotides containing the pelB signal peptide coding sequence. The downstream transcriptional control cassette was generated within intermediate plasmid 131-39.1 by inserting a pair of oligonucleotides containing the pelB signal peptide, Xba I, site, and a ribosome binding site. The oligonucleotides used were:

XbaXho:
5′ CTA GAT ATA ATT AAG GAG ATA AAT ATG AAA TAT CTG CTG CCG ACC GCG GCG GCG GGC CTG CTG CTG CTG GCG GCG CAG CCG GCG ATG GCGC 3′ (SEQ. ID. NO. 15);
and
XhoXba:
5′ TCG AGC GCC ATC GCC GGC TGC GCC GCC AGC AGC AGC AGG CCC GCC GCC GCG GTC GGC AGC AGA TAT TTC ATA TTT ATC TCC TTA ATT ATA T 3′ (SEQ. ID. NO. 16).

The novel pelB leader sequence was optimized for use in E. coli and had the sequence
5′ TAT GAA ATA TCT GCT GCC GAC CGC GGC GGC GGG CCT GCT GCT GCT GGC GGC GCA GCC GGC GAT GGC G 3′ (Seq. ID No. 17).
The resulting plasmid was sequenced to confirm the identity of the insert and digested at the Xbal and XhoI sites to generate a 91 bp fragment which is the downstream transcriptional control cassette.
Construction of pAx131 Vector

The upstream transcriptional control cassette and the downstream transcriptional control cassette were combined with intermediate plasmid p131-44.2 digested with EcoRI and XhoI in a 3-way ligation reaction to produce pAX131 (See FIG. 9). FIG. 10 is a map of the resulting pAX131 vector. The pAX131 was analyzed to determine its nucleic acid sequence (SEQ. ID. NO. 18) which is shown in FIGS. 11A-D.

EXAMPLE 2

Insertion of an alternate upstream transcriptional control cassette

PAX131 vector was digested with Not I restriction enzyme. The resulting DNA overhangs were then filled in with Klenow fragment Polymerase to blunt end the DNA followed by ligation. This was performed to remove the existing Not I site. The Not I deleted PAX131 vector was digested with EcoR I/Xba I, and ligated with a duplexed oligo containing EcoR I and Spe I overhangs (Xba I, and Spe I have compatible ends).

Eco/Spe oligo:
5′ AAT TCA AGG AGT TAA TTA TGA AAA AAA CCG CGA TTG CGA TTG CGG TGG CGC TGG CGG GCT TTG CGA CCG TGG CCC AGG CGG CCT CTA GAA TCT GCG GCC GCA 3′ (SEQ. ID NO. 22)
Spe/Eco oligo:
5′ CTA GTG CGG CCG CAG ATT CTA GAG GCC GCC TGG GCC ACG GTC GCA AAG CCC GCC AGC GCC ACC GCA ATC GCA ATC GCG GTT TTT TTC ATA ATT AAC TCC TTG 3′ (SEQ. ID NO. 23)

The resulting vector (pAX131 Xba/Not) had Xba I, and Not I sites for cloning of a gene, such as light chains, rather than Sac I and Xba I. FIGS. 13A-C show the nucleic acid sequence for vector (pAX131 Xba/Not.

It is contemplated that the present novel vectors can be used in connection with the production and screening of libraries made in accordance with conventional phage display technologies. Both natural and synthetic antibody repertoires have been generated as phage displayed libraries. Natural antibodies can be cloned from B-cell mRNA isolated from peripheral blood lymphocytes, bone marrow, spleen, or other lymphatic tissue of a human or non-human donor. Donors with an immune response to the antigen(s) of interest can be used to create immune antibody libraries. Alternatively, non-immune libraries may be generated from donors by isolating naive antibody B cell genes. PCR using antibody specific primers on the 18^st strand cDNA allows the isolation of light chain and heavy chain antibody fragments which can then be cloned into the display vector.

Synthetic antibodies or antibody libraries can be made up in part or entirely with regions of synthetically derived sequence. Library diversity can be engineered within variable regions, particularly within CDRs, through the use of degenerate oligonucleotides. For example, a single Fab gene may be modified at the heavy chain CDR3 position to contain random nucleotide sequences. The random sequence can be introduced into the heavy chain gene using an oligonucleotide which contains the degenerate coding region in an overlap PCR approach. Alternatively, degenerate oligo cassettes can be cloned into restriction sites that flank the CDR(s) to create diversity. The resulting library generated by this or other approaches can then be cloned into a display vector in accordance with this disclosure.

Upon introduction of the display library into bacteria, phage particles will be generated that have antibody displayed on the surface. The resulting collection of phage-displayed antibodies can be selected for those with the ability to bind to the antigen of interest using techniques known to those skilled in the art. Antibodies identified by this system can be used therapeutically, as diagnostic reagents, or as research tools.

It is contemplated that single and double stranded versions of the vectors described herein are within the scope of the present invention. It is well within the purview of those skilled in the art to prepare either single or double stranded vectors having the features described herein.

It will be understood that various modifications may be made to the embodiments described herein. For example, as those skilled in the art will appreciate, a first gene encoding a fusion protein having an antibody light chain to be fused to and displayed by pVIII and a second gene encoding a heavy chain Fd can be inserted into the vector at the newly created restriction site to provide effective antibody display. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto.

Claims

1. A phagemid vector comprising:

a selectable marker;

a ColE1 origin;

an f1 origin; and

after the ColE1 origin but before the f1 origin, further comprising the following features:

a bacterial transcription terminator;

a promoter,

a first ribosomal binding site;

a first leader sequence;

a first cloning region;

a second ribosomal binding site;

a second leader sequence;

a second cloning region for receiving a gene encoding a polypeptide to be displayed; and

a nucleotide sequence encoding a product that enables display of a polypeptide on the surface of a phagemid particle.

2. A phagemid vector as in claim 1 wherein at least one of the first or second ribosomal binding sites comprises seq. ID No. 13.

3. A phagemid vector as in claim 1 A phagemid vector comprising:

a selectable marker;

a ColE1 origin;

an f1 origin; and

after the ColE1 origin but before the f1 origin, further comprising the following features: a bacterial transcription terminator and a promoter wherein the bacterial transcription terminator is upstream of the promoter;

a first ribosomal binding site;

a first leader sequence;

a first cloning region;

a second ribosomal binding site;

a second leader sequence;

a second cloning region for receiving a gene encoding a polypeptide to be displayed; and

a nucleotide sequence encoding a product that enables display of a polypeptide on the surface of a phagemid particle,

wherein at least one of the first or second leader sequences comprises a sequence selected from the group consisting of seq. ID No. 14 and seq. ID No. 17.

4. A phagemid vector as in claim 1 wherein the nucleotide sequence encoding a product encodes a protein selected from the group consisting of pIII and pVIII.

5. A phagemid vector as in claim 1 wherein the nucleotide sequence encoding a product encodes a truncated pIII.

6. A phagemid vector as in claim 1 wherein the nucleotide sequence encoding a product encodes a synthetic pIII.

7. A phagemid vector as in claim 1 wherein the selectable marker is selected from the group consisting of ampicillin resistance, chloramphenicol transferase resistance, tetracycline resistance and kanamycin resistance.

8. A phagemid vector comprising seq. ID No. 18.

9. A vector comprising a sequence selected from the group consisting of seq. ID Nos. 19, 20 and 21.