Limits...
Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins.

Jacobsson K, Rosander A, Bjerketorp J, Frykberg L - Biol Proced Online (2003)

Bottom Line: From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning.The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies.In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Swedish University of Agricultural Sciences. Box 7025, SE-750 07 UPPSALA. Sweden.

ABSTRACT
Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins.

No MeSH data available.


Related in: MedlinePlus

The pG8SAET-phagemid: The pG8SAET phagemid is a gene VIII-based vector with a universal screening tag, i.e., independently of the ligand used in the panning, putative positive clones can be detected in a colony screening assay by screening for expression of the E-tag.The signal sequence and the E-tag are not in the same reading frame. Instead, insertion of a foreign DNA into either of the cloning sites, NcoI or SnaBI, are required for expression of gene VIII-E-tag-fusion protein. When expressed in a non-supressor strain, protein can be produced without fusion to protein VIII, and recombinant protein purified on a commercially available affinity-column with anti E-tag antibodies (Amersham Biosciences). The complete sequence is available from Genebank AF130864. Primers for sequencing of the inserted DNA:Forward: 5´- TAT CTG GTG GCG TAA CAC CTG CT -3´Reverse: 5´- GAT CGT CAC CCT CGG ATC CCT AGG -3´
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC154567&req=5

Figure 2: The pG8SAET-phagemid: The pG8SAET phagemid is a gene VIII-based vector with a universal screening tag, i.e., independently of the ligand used in the panning, putative positive clones can be detected in a colony screening assay by screening for expression of the E-tag.The signal sequence and the E-tag are not in the same reading frame. Instead, insertion of a foreign DNA into either of the cloning sites, NcoI or SnaBI, are required for expression of gene VIII-E-tag-fusion protein. When expressed in a non-supressor strain, protein can be produced without fusion to protein VIII, and recombinant protein purified on a commercially available affinity-column with anti E-tag antibodies (Amersham Biosciences). The complete sequence is available from Genebank AF130864. Primers for sequencing of the inserted DNA:Forward: 5´- TAT CTG GTG GCG TAA CAC CTG CT -3´Reverse: 5´- GAT CGT CAC CCT CGG ATC CCT AGG -3´

Mentions: In the pG8SAET-vector (Figure 2), the E-tag is in frame with gene VIII, but out of frame with the signal sequence and thus, no expression can be detected. However, insertion of DNA fragments of random length should theoretically restore the reading frame in about one clone in eighteen. Empirically, in a library made in this vector usually 1-2 % of the clones will express the E-tag. After a successful panning, the frequency of clones with an open reading frame from the signal sequence into gene VIII should increase, and subsequently, the frequency of E-tag positive clones should increase. Thus, the frequency of E-tag positive clones is a measure of putative correct clones and is a useful tool for following “specific” enrichment of clones after panning.


Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins.

Jacobsson K, Rosander A, Bjerketorp J, Frykberg L - Biol Proced Online (2003)

The pG8SAET-phagemid: The pG8SAET phagemid is a gene VIII-based vector with a universal screening tag, i.e., independently of the ligand used in the panning, putative positive clones can be detected in a colony screening assay by screening for expression of the E-tag.The signal sequence and the E-tag are not in the same reading frame. Instead, insertion of a foreign DNA into either of the cloning sites, NcoI or SnaBI, are required for expression of gene VIII-E-tag-fusion protein. When expressed in a non-supressor strain, protein can be produced without fusion to protein VIII, and recombinant protein purified on a commercially available affinity-column with anti E-tag antibodies (Amersham Biosciences). The complete sequence is available from Genebank AF130864. Primers for sequencing of the inserted DNA:Forward: 5´- TAT CTG GTG GCG TAA CAC CTG CT -3´Reverse: 5´- GAT CGT CAC CCT CGG ATC CCT AGG -3´
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC154567&req=5

Figure 2: The pG8SAET-phagemid: The pG8SAET phagemid is a gene VIII-based vector with a universal screening tag, i.e., independently of the ligand used in the panning, putative positive clones can be detected in a colony screening assay by screening for expression of the E-tag.The signal sequence and the E-tag are not in the same reading frame. Instead, insertion of a foreign DNA into either of the cloning sites, NcoI or SnaBI, are required for expression of gene VIII-E-tag-fusion protein. When expressed in a non-supressor strain, protein can be produced without fusion to protein VIII, and recombinant protein purified on a commercially available affinity-column with anti E-tag antibodies (Amersham Biosciences). The complete sequence is available from Genebank AF130864. Primers for sequencing of the inserted DNA:Forward: 5´- TAT CTG GTG GCG TAA CAC CTG CT -3´Reverse: 5´- GAT CGT CAC CCT CGG ATC CCT AGG -3´
Mentions: In the pG8SAET-vector (Figure 2), the E-tag is in frame with gene VIII, but out of frame with the signal sequence and thus, no expression can be detected. However, insertion of DNA fragments of random length should theoretically restore the reading frame in about one clone in eighteen. Empirically, in a library made in this vector usually 1-2 % of the clones will express the E-tag. After a successful panning, the frequency of clones with an open reading frame from the signal sequence into gene VIII should increase, and subsequently, the frequency of E-tag positive clones should increase. Thus, the frequency of E-tag positive clones is a measure of putative correct clones and is a useful tool for following “specific” enrichment of clones after panning.

Bottom Line: From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning.The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies.In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Swedish University of Agricultural Sciences. Box 7025, SE-750 07 UPPSALA. Sweden.

ABSTRACT
Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins.

No MeSH data available.


Related in: MedlinePlus