Pressure treatment of tailspike aggregates rapidly produces on‐pathway folding intermediates

Lefebvre, Brian G.; Robinson, Anne S.

doi:10.1002/bit.10607

Cited by 26 publications

(40 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In addition, proteins can aggregate to form net-irreversible species that are not readily dissociable unless one exposes them to extremely high concentrations of chemical denaturants [7,28,29], pressure [30–32], and/or temperature [28]. In these cases, the proteins within the aggregates have lost some or all of their native secondary and/or tertiary structure; doing so allows them to create multiple very strong contacts (e.g., hydrophobic interactions and hydrogen bonds) between proteins by inter-digitating chains from multiple proteins.…”

Section: Why and How Do Proteins Aggregate?mentioning

confidence: 99%

Protein aggregation and its impact on product quality

Roberts

2014

Current Opinion in Biotechnology

243

175

View full text Add to dashboard Cite

Protein pharmaceutical products are typically active as folded monomers that are composed of one or more protein chains, such as the heavy and light chains in monoclonal antibodies that are a mainstay of current drug pipelines. There are numerous possible aggregated states for a given protein, some of which are potentially useful, while most of which are considered deleterious from the perspective of pharmaceutical product quality and performance. This review provides an overview of how and why different aggregated states of proteins occur, how this potentially impacts product quality and performance, fundamental approaches to control aggregate formation, and the practical approaches that are currently used in the pharmaceutical industry.

show abstract

Section: Why and How Do Proteins Aggregate?mentioning

confidence: 99%

Protein aggregation and its impact on product quality

Roberts

2014

Current Opinion in Biotechnology

243

175

View full text Add to dashboard Cite

show abstract

“…A further troubling issue has been the presence of only two detectable bands on SDS-PAGE-the monomer and the trimer-although nondenaturing PAGE consistently showed disulfide-bonded oligomeric species, predominantly as dimers and trimers. The appearance of oligomeric folding intermediates was also observed during size-exclusion chromatography (Lefebvre and Robinson 2003 …”

mentioning

confidence: 91%

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

Kim

Robinson

2006

Protein Science

Self Cite

View full text Add to dashboard Cite

Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.Keywords: P22 tailspike protein (TSP); transient disulfide bond; assembly; disulfide shuffling; SDS P22 tailspike protein is a member of the elongated b-helix family of viral adhesins that participates in polysaccharide binding during viral infection that includes pectate lyase C and pertactin (Mitraki et al. 2002;Simkovsky and King 2006). Tailspike is a homotrimer and each monomer has 666 amino acid residues. Tailspike has four major domains: the N-terminal head-binding domain; the b-helix domain, which is formed by alternating b strands and turns; a bsheet prism domain, where three chains are interdigitated; and the C-terminal domain (Steinbacher et al. 1994(Steinbacher et al. , 1997. Each chain in the native trimer has eight reduced cysteines: six are located in the b-helix domain and two are in the Cterminal domain. Disulfide bonds are formed during tailspike assembly, and they are believed to play critical roles in the registration of the three tailspike chains (Robinson and King 1997;Haase-Pettingell et al. 2001;Danek andRobinson 2003, 2004). Our current model is that intermolecular disulfide bonds form between the C613 on one chain and the C635 on another chain in the C-terminal domain to help align the three subunits. Site-directed mutagenesis of either C613 or C635 significantly decreased the assembly kinetics of tailspike as well as final yields both in vivo and in vitro (Haase-Pettingell et al. 2001;Danek and Robinson 2003). Since the intermolecular disulfide bonds are absent in the native trimer, reduction of those disulfide bonds is a required step in tailspike folding. How does the reduction occur? The nascent chains fold on the ribosome while translation is in progress, and one could imagine many sources of reductants (Brunschier et al. 1993;Gordon et al. 1994;Sather and King 1994;Clark and King 2001). However, in vitro, purified tailspike manages to fold into native trimer in the absence of redox buffers to yields of 80%-90% (Danek and Robinson 2004). A further troubling issue has been the presence of only two detectable bands on SDS-PAGE-the monomer and the trimer-although nondenaturing P...

show abstract

“…One approach applies very high hydrostatic pressures (400 bar) to refold recombinant proteins from inclusion bodies. [13–16] We report a novel method by applying finely controlled levels of shear stress to refold proteins trapped in the inclusion body. This method may be capable of broadening the utility of bacterial over-expression, and could transform industrial and research production of proteins.…”

mentioning

confidence: 99%

Shear‐Stress‐Mediated Refolding of Proteins from Aggregates and Inclusion Bodies

et al. 2015

View full text Add to dashboard Cite

Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of the much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is >100-times faster than conventional, overnight dialysis. This rapid refolding technique could significantly shorten the times, lower costs, and reduce the waste streams associated with protein expression for a wide-range of industrial and research applications.

show abstract

Pressure treatment of tailspike aggregates rapidly produces on‐pathway folding intermediates

Cited by 26 publications

References 39 publications

Protein aggregation and its impact on product quality

Protein aggregation and its impact on product quality

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

Shear‐Stress‐Mediated Refolding of Proteins from Aggregates and Inclusion Bodies

Contact Info

Product

Resources

About