Expression and Purification of Uniformly 15N-Labeled Amyloid β Peptide 1-40 in Escherichia coli

Abstract

For biophysical studies using heteronuclear NMR analysis of amyloid beta peptide, construction of an efficient and high yield expression system of uniformly isotopic labeled amyloid beta peptide is desirable. Here we succeeded in obtaining (15)N-labeled amyloid beta 1-40 expressed by attachment to hen egg white lysozyme as a fusion protein.

A routine method for cloning, expressing and purifying Aβ(1–42) for structural NMR studies

“…Figure 4 displays a well-resolved HSQC spectrum of monomeric Aβ(1-42). Cross-peak positions are well-matched to previous spectra of either or Aβ(1-42) with similar buffer composition and temperature (Hou et al 2004;Long et al 2011;Nagata-Uchiyama et al 2007;Williamson et al 2006;Yan et al 2008;Danielsson et al 2006;Broersen et al 2011;Rezaei-Ghaleh et al 2011;Ghalebani et al 2012). In addition, samples maintained at 5 °C were stable for periods exceeding 1 week, which allowed confirmation of 1 H 15 N HSQC assignments through a longer triple-resonance 15 N-edited HNCACB experiment.…”

“…Resonances for Ala 2 and Asp 7 in HSQC spectra, overlapped with Ala 30 and Asp 23 , respectively, were assigned directly by reference to Williamson et al (2006); however, it should be noted that our experiments could not confirm this as neither the HN(i)/CαCβ(i-1) or HN(i)/CαCβ(i) correlation was observed in HNCACB spectra for these residues. In other studies, absence of His 13 and His 14 , and deviations of Asp 7 , Asn 27 and Lys 28 amide cross-peaks positions, relative to our HSQC spectra also exist (Hou et al 2004;Long et al 2011;Nagata-Uchiyama et al 2007;Danielsson et al 2006), and may possibly be explained by differences in sample preparation (i.e., our inclusion of NaCl, and strength of ammonia pretreatment). Furthermore, the Met 35 chemical shift position suggests that Aβ(1-42) is in the reduced form (Hou et al 2004), consistent with ESI-MS characterization (Fig.…”

“…Firstly, despite being considerably more soluble than Aβ(1-42), the fusion construct still localizes primarily in inclusion bodies (Satakarni and Curtis 2011). While this has commonly been observed for other fusion constructs (Finder et al 2010;Long et al 2011;Nagata-Uchiyama et al 2007;Shahnawaz et al 2007), requirements for dissolution into denaturing buffers is undesirable owing to risks of protein modifications (Kollipara and Zahedi 2013) and losses in yields and time associated with refolding. Fortunately, SUMO-Aβ(1-42) could be refolded by onresin buffer exchange, effectively averting days of dialysis.…”

“…In this approach, recombinant DNA techniques are used to create a fusion protein where a well-behaved protein subunit is both affinity-tagged for purification ease and linked to a protein of interest with a peptide linker plus protease cleavage site, such as for TEV, Factor Xa, or enterokinase. While Aβ(1-40) and Aβ(1-42) free of extraneous N-terminal methionine (as is often the case for straight biosynthesis in E. coli) or other residues from sloppy protease cleavage has apparently been produced by this approach (Finder et al 2010;Hortschansky et al 2005;Long et al 2011;Nagata-Uchiyama et al 2007), adaptations of these methods for isotope labeling have only been comprehensively described for the less-amyloidogenic (and less disease-relevant) Aβ(1-40), either providing low (<2 mg/L of culture) or undisclosed final yields (Nagata-Uchiyama et al 2007). This may in part be due to the typical losses in yield owing to the use of specialized minimal growth media compared to high-density media for routine biosynthetic protein production at natural isotopic abundance (Long et al 2011).…”

Chemical Strategies for Controlling Protein Folding and Elucidating the Molecular Mechanisms of Amyloid Formation and Toxicity

“…98 To overcome this limitation, some groups have investigated the expression of the Aβ peptide or its fragments as fusion proteins for NMR studies. [175][176][177] While the yields increased dramatically, these strategies require the placement of protease cleavage sites between the Aβ domain and the fusion partner, which leaves additional residues after its removal, thus introducing potential artifacts in the structure and aggregation properties of those peptides.…”