Dataset Information

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis.

ABSTRACT: Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a heterobifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences ("helping hands") onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ?-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebola virus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic l-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of d-GroES, which was inactive in a heterochiral mixture with recombinant GroEL, providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.

SUBMITTER: Jacobsen MT

PROVIDER: S-EPMC5094862 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis.

Jacobsen Michael T MT Petersen Mark E ME Ye Xiang X Galibert Mathieu M Lorimer George H GH Aucagne Vincent V Kay Michael S MS

Journal of the American Chemical Society 20160901 36

Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a new general tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a he ...[more]

PMID: 27532670

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Project description:BackgroundA growing number of Americans are enrolled in high-deductible health plans (HDHPs). Enrollees in HDHPs, particularly those with chronic conditions, face high out-of-pocket costs and often delay or forgo needed care owing to cost. These challenges could be mitigated by the use of cost-conscious strategies when seeking health care, such as discussing costs with providers, saving for medical expenses, and using web-based tools to compare prices, but few HDHP enrollees engage in such cost-conscious strategies. A novel behavioral intervention could enable HDHP enrollees with chronic conditions to adopt these strategies, but it is unknown which intervention features would be most valued and used by this patient population.ObjectiveThis study aimed to assess preferences among HDHP enrollees with chronic conditions for a novel behavioral intervention that supports the use of cost-conscious strategies when planning for and seeking health care.MethodsIn an exploratory sequential mixed methods study among HDHP enrollees with chronic conditions, we conducted 20 semistructured telephone interviews and then surveyed 432 participants using a national internet survey panel. Participants were adult HDHP enrollees with diabetes, hypertension, coronary artery disease, chronic obstructive pulmonary disease, or asthma. The interviews and survey assessed participants' health care experiences when using HDHPs and their preferences for the content, modality, and frequency of use of a novel intervention that would support their use of cost-conscious strategies when seeking health care.ResultsApproximately half (11/20, 55%) of the interview participants reported barriers to using cost-conscious strategies. These included not knowing where to find information and worrying that the use of cost-conscious strategies would be very time consuming. Most (18/20, 90%) interviewees who had discussed costs with providers, saved for medical expenses, or used web-based price comparison tools found these strategies to be helpful for managing their health care costs. Most (17/20, 85%) interviewees expressed interest in an intervention delivered through a website or phone app that would help them compare prices for services at different locations. Survey participants were most interested in learning to compare prices and quality, followed by discussing costs with their providers and putting aside money for care, through a website-based or email-based intervention that they would use a few times a year.ConclusionsRegular use of cost-conscious strategies could mitigate financial barriers faced by HDHP enrollees with chronic conditions. Interventions to encourage the use of cost-conscious strategies should be delivered through a web-based modality and focus on helping these patients in navigating their HDHPs to better manage their out-of-pocket spending.

Dataset Information

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis.

Publications

A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis.

Similar Datasets

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