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pSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins.
Protein Expr Purif. 1999 Jun; 16(1):63-9.PE

Abstract

The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli PhoA/S gene that encodes an alkaline phosphatase with increased specific activity. The restriction sites incorporated into pSKAP/S allowed the scFv genes to be easily transferred from pUC119-derived phagemid vectors that are used frequently in phage display antibody library technology. Strong transcriptional control of expression was achieved using the tetracycline promoter, and induction of different individual clones with anhydrotetracycline resulted in secretion of most of the scFv-alkaline phosphatase fusion proteins into the culture medium. Although some of the clones secreted fusion proteins that were retained in the periplasm, these proteins could be isolated with a simple extraction procedure. Increased amounts of a scFv-alkaline phosphatase fusion protein were obtained when expressed in the pSKAP/S vector compared with expression in a vector incorporating the lac promoter. Testing for binding of the scFv-alkaline phosphatase fusion proteins to antigen was possible in an ELISA without the need for additional enzyme-conjugated antibodies. The pSKAP/S vector was successfully used to obtain scFv fragments from a preparation of phage-antibody clones after subcloning and expression of individual clones as scFv-alkaline phosphatase fusions, whereas fewer clones (and clones with different properties) were obtained from the same phage-antibody preparations when expressed as soluble scFv fragments. Therefore, the pSKAP/S vector was shown to be useful in extending the range of scFv obtained from phage display libraries.

Authors+Show Affiliations

Laboratory for Monoclonal Antibodies, Wageningen Agricultural University, Wageningen, 6700 ES, The Netherlands.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10336861

Citation

Griep, R A., et al. "PSKAP/S: an Expression Vector for the Production of Single-chain Fv Alkaline Phosphatase Fusion Proteins." Protein Expression and Purification, vol. 16, no. 1, 1999, pp. 63-9.
Griep RA, van Twisk C, Kerschbaumer RJ, et al. PSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins. Protein Expr Purif. 1999;16(1):63-9.
Griep, R. A., van Twisk, C., Kerschbaumer, R. J., Harper, K., Torrance, L., Himmler, G., van der Wolf, J. M., & Schots, A. (1999). PSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins. Protein Expression and Purification, 16(1), 63-9.
Griep RA, et al. PSKAP/S: an Expression Vector for the Production of Single-chain Fv Alkaline Phosphatase Fusion Proteins. Protein Expr Purif. 1999;16(1):63-9. PubMed PMID: 10336861.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - pSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins. AU - Griep,R A, AU - van Twisk,C, AU - Kerschbaumer,R J, AU - Harper,K, AU - Torrance,L, AU - Himmler,G, AU - van der Wolf,J M, AU - Schots,A, PY - 1999/5/25/pubmed PY - 1999/5/25/medline PY - 1999/5/25/entrez SP - 63 EP - 9 JF - Protein expression and purification JO - Protein Expr. Purif. VL - 16 IS - 1 N2 - The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli PhoA/S gene that encodes an alkaline phosphatase with increased specific activity. The restriction sites incorporated into pSKAP/S allowed the scFv genes to be easily transferred from pUC119-derived phagemid vectors that are used frequently in phage display antibody library technology. Strong transcriptional control of expression was achieved using the tetracycline promoter, and induction of different individual clones with anhydrotetracycline resulted in secretion of most of the scFv-alkaline phosphatase fusion proteins into the culture medium. Although some of the clones secreted fusion proteins that were retained in the periplasm, these proteins could be isolated with a simple extraction procedure. Increased amounts of a scFv-alkaline phosphatase fusion protein were obtained when expressed in the pSKAP/S vector compared with expression in a vector incorporating the lac promoter. Testing for binding of the scFv-alkaline phosphatase fusion proteins to antigen was possible in an ELISA without the need for additional enzyme-conjugated antibodies. The pSKAP/S vector was successfully used to obtain scFv fragments from a preparation of phage-antibody clones after subcloning and expression of individual clones as scFv-alkaline phosphatase fusions, whereas fewer clones (and clones with different properties) were obtained from the same phage-antibody preparations when expressed as soluble scFv fragments. Therefore, the pSKAP/S vector was shown to be useful in extending the range of scFv obtained from phage display libraries. SN - 1046-5928 UR - https://www.unboundmedicine.com/medline/citation/10336861/pSKAP/S:_An_expression_vector_for_the_production_of_single_chain_Fv_alkaline_phosphatase_fusion_proteins_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1046-5928(99)91041-0 DB - PRIME DP - Unbound Medicine ER -