Single-cell Characterization of Autotransporter-mediated Escherichia coli Surface Display of Disulfide Bond-containing Proteins

Abstract

Background:The ability of autotransporter (AT) to translocate polypeptides with multiple disulfide bonds is controversial. Results: Surface display of functional chymotrypsin (4 S-S) and M18 scFv (2 S-S) was quantitatively characterized. Conclusion: Surface display of functional recombinant protein with multiple disulfide bonds can be achieved using AT system. Significance: Displaying recombinant proteins with disulfide bonds enhances utility of ATs.

Of linkers and autochaperones: an unambiguous nomenclature to identify common and uncommon themes for autotransporter secretion

“…Adding to this ambiguity, other studies define translocator or β-domain as the minimal C-terminal portion of the AT protein required for OM translocation of the passenger. By this definition, the translocator or β-domain always includes the β-barrel domain and the α-helix of the linker but often extends further to include the disordered portion of the linker (Maurer et al, 1999; Oliver et al, 2003a; Ramesh et al, 2012; Sevastsyanovich et al, 2012; Suzuki et al, 1995; Yang et al, 2004). In some cases, the translocator extends still further, well into the folded domain of the passenger (Fischer et al, 2001; Ivie et al, 2010; Velarde and Nataro, 2004).…”

“…For example, a stable core has been found in pertactin (Prn) (Junker et al, 2006), Pet (Renn and Clark, 2008) and IcsA (Kühnel and Diezmann, 2011), and is known to be absent in YapV (Besingi et al, 2013), but its existence has not been tested in any other AT protein. Conversely, the minimal translocator unit has never been determined for pertactin or Hbp, but is well characterized for IgAP (Klauser et al, 1993), Ag43 (Ramesh et al, 2012), IcsA (Suzuki et al, 1995), and AIDA-1 (Maurer et al, 1999). As a result of the limited results available, direct mapping of functional features onto structural units across the diverse AT family is challenging and subject to bias.…”

“…Inclusion of an autochaperone is predicated on it being experimentally shown to rescue folding-deficient passenger mutants when supplied in trans . Translocators correspond to the minimal units required to secrete a passenger, typically a heterologous one. a Charles et al (1989); Emsley et al (1996); Junker et al (2009, 2006); Makoff et al (1990) b Oliver et al (2003a,b); Zhai et al (2011) c Barnard et al (2007, 2012); Brockmeyer et al (2009); Dutta et al (2003); Khan et al (2011); Skillman et al (2005); Szabady et al (2005); Velarde and Nataro (2004) d Otto et al (2005); Saurí et al (2011); Soprova et al (2010); Tajima et al (2010) e Dutta et al (2003); Meza-Aguilar et al (2014); Renn et al (2012); Renn and Clark (2008) f Fink et al (2001); Meng et al (2011) g Johnson et al (2009); Klauser et al (1993, 1990); Pohlner et al (1987, 1995); van Ulsen et al (2003) h Côté and Mourez (2011); Lindenthal and Elsinghorst (1999) i Brandon and Goldberg (2001); Kühnel and Diezmann (2011); May and Morona (2008); Suzuki et al (1995); Teh and Morona (2013) j Heras et al (2014); Ramesh et al (2012); van der Woude and Henderson (2008) k Benz and Schmidt (1992); Berthiaume et al (2007); Charbonneau et al (2006); Charbonneau and Mourez (2007); Gawarzewski et al (2014); Maurer et al (1999); Rutherford et al (2006); Suhr et al (1996) l Besingi et al (2013) m van den Berg (2010); Wilhelm et al (2007, 199...…”

Functional Enrichment by Direct Plasmid Recovery After Fluorescence Activated Cell Sorting

“…In the present study, we have quantitatively compared a direct plasmid recovery method with the more traditional method of culturing sorted cells following FACS, focusing on maximizing the yield of the sub-populations expressing the desired phenotype. We demonstrate enrichment and recovery using two disparate protein systems: E. coli MC1061 cells expressing the cytotoxic protease chymotrypsin, and E. coli HF19 cells expressing variants of the AraC regulatory protein that activate expression of GFP (8, 9). In both systems, direct plasmid recovery was found to be superior in comparison to recovery by culturing sorted cells.…”

“…Buffer 3 contains 0.1 M NaOH, 0.7% SDS, 0.7 mM EDTA, and 1% isopropanol (10). For evaluating buffers 1 and 2, E. coli MC1061 cells (N 0 = 50,000 in ~200 μL) harboring plasmid pBAD_AChy_700 (p15A origin, 15–20 copies/cell) were sorted and incubated with an equal volume of 2X lysis buffer at 25 °C for 15 min (8, 11). Plasmids were purified using Zymo DNA clean and concentrator kit (Zymo research, CA).…”