Chromosomal His‐tagging: An alternative approach to membrane protein purification

Abstract

Membrane proteins are of keen interest to structural biologists, as they are known to act as receptors, adhesins, sensors, transporters, and signal-transducers of living cells. During the past few decades, the efforts made to study the bacterial membrane proteins have been impaired by the problems encountered during the production and purification of native proteins. Herein we demonstrate that the Campylobacter jejuni CadF protein, which was isolated using a novel purification strategy, exhibits biological activity as evidenced by channel activity in lipid bilayers. CadF, an E. coli OmpA-like protein, facilitates the binding of C. jejuni to the extracellular matrix component, fibronectin.

Expression patterns and role of the CadF protein inCampylobacter jejuniandCampylobacter coli

“…Using two CadF-specific antisera, a 37 kDa band (p37) and a less-prominent 32 kDa band (p32) were detected in C. jejuni strains by immunoblotting of total cell lysates. These bands corresponded to previously described CadF proteins (Konkel et al, 1997;Mamelli et al, 2006Mamelli et al, , 2007. While p37 was present in all C. jejuni isolates, five human isolates and one from a calf failed to exhibit the lessprominent p32 band (Table 1).…”

“…While p37 was detected in all C. jejuni isolates of human and animal origin, the less-prominent p32 band was found only in 62% of the C. jejuni isolates of human origin and in 96% of the C. jejuni isolates of animal origin. Heat modifiablity is a well-known feature of membrane proteins (Nakamura & Mizushima, 1976;Bolla et al, 1995), including CadF (Konkel et al, 1997(Konkel et al, , 1999bMamelli et al, 2006Mamelli et al, , 2007. Therefore, the migration of CadF as two protein species is likely caused by their heatmodifiable conformational state, where p32 is the incompletely denaturated and partially folded form of CadF.…”

In silico Analysis of CadF epitope- based vaccine design against Campylobacter jejuni

“…and is belonging to the superfamily of outer member proteins (ompA). [28][29][30]. In addition, our research also showed the correct topology model based on phyre2 server that predicts CadF is a stable target.…”

Validation of a Burkholderia pseudomallei Hypothetical Protein and Determination of Its Translational Start Codon Using Chromosomal Integration of His-Tag Coding Sequence

“…Furthermore, the purified native form of protein BPSS1356 obtained from its original host is more suitable for observation compared to its recombinant counterpart produced from a heterologous expression system in another species such as E. coli. The chromosomal His-tagging strategy was previously shown to be effective in the purification of biologically active proteins such as ribonucleoprotein complex from E. coli [6], membrane protein from Campylobacter jejuni [8] and RNA polymerase from Bacillus subtilis [2]. This is the first demonstration that the same strategy can be applied to validate a hypothetical protein in B. pseudomallei by purifying the His-tagged protein for analysis to determine its identity.…”