About: High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli

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About: High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli Goto Sponge NotDistinct Permalink

An Entity of Type : schema:ScholarlyArticle, within Data Space : wasabi.inria.fr associated with source document(s)

Attributes	Values
type	Academic Article research paper schema:ScholarlyArticle
isDefinedBy	Covid-on-the-Web dataset
has title	High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli
Creator	Lin, Zhihua Lei, Huanzong Ye, Tingmei
Source	Medline; PMC
abstract	Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO–scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO–scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.
has issue date	2008-11-01(xsd:dateTime)
bibo:doi	10.1007/s00253-008-1655-3
bibo:pmid	18795288
has license	no-cc
sha1sum (hex)	b99ef4f30ca8cc31ddcfaf337985bee483e23092
schema:url	https://doi.org/10.1007/s00253-008-1655-3
resource representing a document's title	High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli
has PubMed Central identifier	PMC7079844
has PubMed identifier	18795288
schema:publication	Appl Microbiol Biotechnol
resource representing a document's body	covid:b99ef4f30ca8cc31ddcfaf337985bee483e23092#body_text
is schema:about of	named entity 'VEGF165' named entity 'SMALL' named entity 'fusion protein' named entity 'cell proliferation' named entity 'highly' named entity 'protease' named entity 'problem' named entity 'scFv' named entity 'single-chain variable fragment' named entity 'FUSED' named entity 'ANTIBODIES' named entity 'CLASS' named entity 'EXPRESSION' named entity 'THERAPEUTICS' named entity 'ENVIRONMENT' named entity 'STRAIN' named entity 'ARTIFICIAL' named entity 'FUSION PROTEIN' named entity 'INTRACELLULAR' named entity 'EASY' named entity 'CURRENTLY' named entity 'CHALLENGE' named entity 'ALLOWS' named entity 'RECOMBINANT ANTIBODIES' named entity 'EXPRESSION LEVEL' named entity 'IN VITRO ACTIVITY' named entity 'CONTAINS' named entity 'UP TO' named entity 'UBIQUITIN-RELATED' named entity 'IS A' named entity 'CLEAVED' named entity 'ANALYSIS' named entity 'MODIFIER GENE' named entity 'PROBLEM' named entity 'PURITY' named entity 'TIME-OF-FLIGHT MASS SPECTROSCOPY' named entity 'SINGLE-CHAIN ANTIBODY' named entity 'DIFFICULT' named entity 'SEQUENCING' named entity 'SPECIFIC' named entity 'PROTEASE' named entity 'HIGH YIELD' named entity 'N-TERMINAL AMINO ACID' named entity 'NATIVE' named entity 'SUCCESSFUL' named entity 'ASSISTED' named entity 'FUSION GENE' covid:arg/b99ef4f30ca8cc31ddcfaf337985bee483e23092 named entity 'SINGLE-CHAIN VARIABLE FRAGMENT' named entity 'OPTIMAL' named entity 'PRODUCTION' named entity 'VEGF165' named entity 'NTA' named entity 'DERIVED' named entity 'REAL' named entity 'INDUCED' named entity 'AFFINITY CHROMATOGRAPHY' named entity 'STUDY' named entity 'UMBILICAL VEIN' named entity 'DISULFIDE BONDS' named entity 'ANTIBODY FRAGMENT' named entity 'OBTAIN' named entity 'PRESENTED' named entity 'ACTIVE' named entity 'CELLULAR' named entity 'PROTEIN ' named entity 'STRATEGY' named entity 'SMALL' named entity 'TO FORM' named entity 'BACTERIAL' named entity 'CONFIRMED BY' named entity 'ENDOTHELIAL CELL PROLIFERATION' named entity 'TOTAL' named entity 'HUMAN' named entity 'MATRIX' named entity 'FUSION' named entity 'CHARACTERIZATION' named entity 'SUMO' named entity 'PROTEIN'

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