Tags

Type your tag names separated by a space and hit enter

A simple vector system to improve performance and utilisation of recombinant antibodies.
BMC Biotechnol 2006; 6:46BB

Abstract

BACKGROUND

Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression.

RESULTS

We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs). Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3). The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2 (DE3)) was investigated and found to be inferior to periplasmic expression in BL21 (DE3) cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner), bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule), or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and immunohistochemistry.

CONCLUSION

Nine scFv expression vectors have been generated and tested. Three vectors showed utility for expression of functional scFv fragments. One vector, pSANG14-3F, produces scFv-alkaline phosphatase fusion molecules which offers a simple, convenient and sensitive way of determining the reactivity of recombinant antibody fragments in a variety of common assay systems.

Authors+Show Affiliations

Atlas of Protein Expression, Wellcome Trust Sanger Institute, Genome Campus, Morgan Building, Hinxton, Cambridgeshire, CB10 1HH, UK. cecile.d.martin@gsk.com <cecile.d.martin@gsk.com>No 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

17156422

Citation

Martin, Cecile D., et al. "A Simple Vector System to Improve Performance and Utilisation of Recombinant Antibodies." BMC Biotechnology, vol. 6, 2006, p. 46.
Martin CD, Rojas G, Mitchell JN, et al. A simple vector system to improve performance and utilisation of recombinant antibodies. BMC Biotechnol. 2006;6:46.
Martin, C. D., Rojas, G., Mitchell, J. N., Vincent, K. J., Wu, J., McCafferty, J., & Schofield, D. J. (2006). A simple vector system to improve performance and utilisation of recombinant antibodies. BMC Biotechnology, 6, p. 46.
Martin CD, et al. A Simple Vector System to Improve Performance and Utilisation of Recombinant Antibodies. BMC Biotechnol. 2006 Dec 7;6:46. PubMed PMID: 17156422.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A simple vector system to improve performance and utilisation of recombinant antibodies. AU - Martin,Cecile D, AU - Rojas,Gertrudis, AU - Mitchell,Joanne N, AU - Vincent,Karen J, AU - Wu,Jiahua, AU - McCafferty,John, AU - Schofield,Darren J, Y1 - 2006/12/07/ PY - 2006/08/24/received PY - 2006/12/07/accepted PY - 2006/12/13/pubmed PY - 2006/12/29/medline PY - 2006/12/13/entrez SP - 46 EP - 46 JF - BMC biotechnology JO - BMC Biotechnol. VL - 6 N2 - BACKGROUND: Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. RESULTS: We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs). Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3). The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2 (DE3)) was investigated and found to be inferior to periplasmic expression in BL21 (DE3) cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner), bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule), or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and immunohistochemistry. CONCLUSION: Nine scFv expression vectors have been generated and tested. Three vectors showed utility for expression of functional scFv fragments. One vector, pSANG14-3F, produces scFv-alkaline phosphatase fusion molecules which offers a simple, convenient and sensitive way of determining the reactivity of recombinant antibody fragments in a variety of common assay systems. SN - 1472-6750 UR - https://www.unboundmedicine.com/medline/citation/17156422/A_simple_vector_system_to_improve_performance_and_utilisation_of_recombinant_antibodies_ L2 - https://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-6-46 DB - PRIME DP - Unbound Medicine ER -