TY - JOUR
T1 - Purification of recombinant high molecular weight two-partner secretion proteins from Escherichia coli
AU - Fleckenstein, James M.
AU - Roy, Koushik
N1 - Funding Information:
ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (National Center for Research Resources) RR16190-05, the Department of Veterans Affairs and funds from the University of Tennessee Microbial Pathogenesis Research Center.
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
UR - https://www.scopus.com/pages/publications/68149170675
U2 - 10.1038/nprot.2009.87
DO - 10.1038/nprot.2009.87
M3 - Article
C2 - 19707189
AN - SCOPUS:68149170675
SN - 1754-2189
VL - 4
SP - 1083
EP - 1092
JO - Nature Protocols
JF - Nature Protocols
IS - 7
ER -