title: Purification of recombinant high molecular weight two-partner secretion proteins from Escherichia coli.
abstract: This protocol describes the purification of a recombinant high molecular weight (HMW) two-partner secretion exoprotein (generically referred to as TpsA proteins) from Escherichia coli using methods developed recently to obtain highly purified flagellin-free recombinant EtpA (rEtpA) glycoprotein. The protocol addresses problems frequently encountered with the expression of these HMW proteins, namely plasmid instability and protein degradation, as well as a recently recognized issue of flagellin contamination. Briefly, the TpsA protein of interest is expressed with its outer membrane transporter (TpsB) protein in a flagellin-minus recombinant E. coli background. Culture supernatants are collected, concentrated through high molecular weight cutoff filters, followed by purification by size exclusion column chromatography. Details are included for the expression of HMW TpsA glycoproteins as polyhistidine-tagged molecules, which can be further purified by metal affinity chromatography (MAC). Using this protocol, it is possible to obtain highly purified microgram-milligram quantities of the TpsA protein of interest within 2-3 days.
authors.affiliation: Department of Veterans Affairs Medical Center Research (151), Memphis, Tennessee, USA. jflecke1@tennessee.edu
authors.forename: James M
authors.initials: JM
authors.lastname: Fleckenstein
authors.forename: Koushik
authors.initials: K
authors.lastname: Roy
mesh_terms: D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins
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