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A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

Xiao X, Chen Y, Mugabe S, Gao C, Tkaczyk C, Mazor Y, Pavlik P, Wu H, Dall'Acqua W, Chowdhury PS - PLoS ONE (2015)

Bottom Line: There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output.When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium.Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Antibody Discovery and Protein Engineering, MedImmune, LLC., Gaithersburg, MD, 20878, United States of America.

ABSTRACT
High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

No MeSH data available.


Related in: MedlinePlus

scFv.Fcs expressed in mammalian cells are biologically active.Opsonophagocytosis activity of scFv.Fc fusion proteins for clone 1, clone 2 and negative control clone in supernatants from 293F cells transfected with the respective pSplicev.5 plasmid. Black and white bars represent OPK activity of scFv.Fc at 10 μg/ml and 1 μg/ml, respectively. The mean values for each group are derived from experiments done in triplicates and the s.d. is represented by error bars.
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pone.0140691.g006: scFv.Fcs expressed in mammalian cells are biologically active.Opsonophagocytosis activity of scFv.Fc fusion proteins for clone 1, clone 2 and negative control clone in supernatants from 293F cells transfected with the respective pSplicev.5 plasmid. Black and white bars represent OPK activity of scFv.Fc at 10 μg/ml and 1 μg/ml, respectively. The mean values for each group are derived from experiments done in triplicates and the s.d. is represented by error bars.

Mentions: Supernatants of HEK293F cells transfected with pSplice v.5 of clones 1 and 2 as well as a negative control scFv.Fc were tested for opsonophagocytosis (OPK) activity which is a function of the Fc segment. As shown in Fig 6, clones 1 and 2 but not a negative control scFv.Fc sample showed potent OPK activity. This indicates that pSplice vector expresses fusion proteins in HEK293F cells that can be tested for Fc based biological activities.


A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

Xiao X, Chen Y, Mugabe S, Gao C, Tkaczyk C, Mazor Y, Pavlik P, Wu H, Dall'Acqua W, Chowdhury PS - PLoS ONE (2015)

scFv.Fcs expressed in mammalian cells are biologically active.Opsonophagocytosis activity of scFv.Fc fusion proteins for clone 1, clone 2 and negative control clone in supernatants from 293F cells transfected with the respective pSplicev.5 plasmid. Black and white bars represent OPK activity of scFv.Fc at 10 μg/ml and 1 μg/ml, respectively. The mean values for each group are derived from experiments done in triplicates and the s.d. is represented by error bars.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4607404&req=5

pone.0140691.g006: scFv.Fcs expressed in mammalian cells are biologically active.Opsonophagocytosis activity of scFv.Fc fusion proteins for clone 1, clone 2 and negative control clone in supernatants from 293F cells transfected with the respective pSplicev.5 plasmid. Black and white bars represent OPK activity of scFv.Fc at 10 μg/ml and 1 μg/ml, respectively. The mean values for each group are derived from experiments done in triplicates and the s.d. is represented by error bars.
Mentions: Supernatants of HEK293F cells transfected with pSplice v.5 of clones 1 and 2 as well as a negative control scFv.Fc were tested for opsonophagocytosis (OPK) activity which is a function of the Fc segment. As shown in Fig 6, clones 1 and 2 but not a negative control scFv.Fc sample showed potent OPK activity. This indicates that pSplice vector expresses fusion proteins in HEK293F cells that can be tested for Fc based biological activities.

Bottom Line: There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output.When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium.Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Antibody Discovery and Protein Engineering, MedImmune, LLC., Gaithersburg, MD, 20878, United States of America.

ABSTRACT
High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

No MeSH data available.


Related in: MedlinePlus