RRC ID 61150
Author Iwaki T, Hara K, Umemura K.
Title Nanobody production can be simplified by direct secretion from Escherichia coli.
Journal Protein Expr Purif
Abstract It is well known that camelids (camels and llamas) have fully functional antibodies with only a heavy chain consisting of a single variable domain and two constant domains. This single variable domain is called a "nanobody" and many nanobodies are synthesized in the cytosol of Escherichia coli, however, most of the nanobodies become inclusion bodies without tags to enhance their solubility. We generated a vector system to enable the secretary expression of nanobodies in Escherichia coli. In this system, several NBs were secreted into the culture supernatant. Since the vector contained 6xHis tag and AviTAG, biotinylation (even fluorescent-labeled) of AviTAG was achieved during cell culture, and purification of the supernatant was a step by immobilized metal ion adsorption chromatography. The procedure described in this study is believed to be as simple as regular plasmid minipreps. Therefore, many laboratories can use this method.
Volume 170
Pages 105607
Published 2020-6-1
DOI 10.1016/j.pep.2020.105607
PII S1046-5928(20)30045-0
PMID 32062022
MeSH Animals Avidin / chemistry Biotinylation Camelids, New World Camelus Chromatography, Affinity Cloning, Molecular Culture Media, Conditioned / chemistry Enzyme-Linked Immunosorbent Assay Escherichia coli / genetics Escherichia coli / metabolism* Gene Expression Histidine / genetics Histidine / metabolism Oligopeptides / genetics Oligopeptides / metabolism Plasmids / chemistry Plasmids / metabolism* Recombinant Proteins / genetics Recombinant Proteins / isolation & purification Recombinant Proteins / metabolism Single-Domain Antibodies / genetics Single-Domain Antibodies / isolation & purification* Single-Domain Antibodies / metabolism
IF 1.513
DNA material pMAK461 (RDB18226) pMAK462 (RDB18227) pMAK463 (RDB18228) pMAK464 (RDB18229)