Single-Shot Flow Synthesis of D-Proteins for Mirror-Image Phage Display

Callahan, Alex J.; Gandhesiri, Satish; Travaline, Tara L.; Salazar, Lia Lozano; Hanna, Stephanie; Lee, Yen‐Chun; Li, Kunhua; Tokareva, Olena; Swiecicki, Jean-Marie; Loas, Andrei; Verdine, Gregory L.; McGee, John H.; Pentelute, Bradley L.

doi:10.26434/chemrxiv-2023-x86xp

Cited by 11 publications

(13 citation statements)

References 52 publications

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“…Points from the average of three technical replicates are plotted (see Table S2). ERG from folding selection also displays a melting temperature (T M ) determined by variable temperature CD that closely matches the literature-reported value for recombinant protein 24 . Points are from the result of a single replicate (see Table S3).…”

Section: Synthetic Side Products Misfoldsupporting

confidence: 80%

“…Analytical RP-HPLC and LC-MS spectra of the purified protein are shown in Figure 2E. The circular dichroism spectrum of the synthetic ERG matches that of ERG from recombinant expression and previously reported AFPS synthesis 24 (Figure 2F), suggesting it is the native structure.…”

Section: Folding Selections Afford Purified Synthetic Proteinssupporting

confidence: 79%

“…F) ERG from folding selection displays the expected circular dichroism (CD) spectra. A CD spectrum was recorded from 195 to 260 nm of ERG from Folding Selection, displaying an alpha-helical structure similar to that of recombinant ERG 24 . Points from the average of three technical replicates are plotted (see Table S2).…”

Section: Synthetic Side Products Misfoldmentioning

confidence: 99%

“…The DNA binding domain of BCL11A comprises three tandem C2H2 zinc fingers 37 , and we anticipated that it would be a challenging target for folding selection due to its structural complexity. We synthesized the protein with AFPS with its three methionine residues substituted for the non-natural norleucine (Nle), as previously reported 24 , due to their sensitivity to oxidation. Despite including non-natural amino acids, a HIC protocol outlined in Figure 5A afforded 20 g of purified BCL11A (Figure 5 A, extrapolating to 0.2 mg from processing the full synthesis, SI section 5.4).…”

Section: Diversification Of Folding Selection Protocols Affords a Var...mentioning

confidence: 99%

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Same Day Access to Proteins Enabled by Folding Selections

Callahan

Rondon

Salazar

et al. 2023

Preprint

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From laundry detergent to biomedicines, proteins are core components of modern technology. However, despite significant progress, modern recombinant protein expression still requires days to months to deliver pure, functional material. Chemical protein synthesis can also afford proteins for study with similar timeframes at the chemistry stage, with additional time dedicated post-synthesis for handling as separation of the numerous closely related side-products requires weeks to months. To overcome these rate-limiting barriers, we propose folding selection as a new framework to understand synthetic side products and enable rapid purification. We demonstrate that the minor chemical modifications present on synthetic side products result in substantially altered physical-chemical properties and that simple bio-purification techniques can separate them from the native protein in hours. With this strategy, we demonstrate the production of nine functional synthetic proteins in under ten hours each, including disulfide-containing enzymes and transcription factor domains with non-natural amino acids. Furthermore, only possible with this technology, we access homogeneous, post-translationally modified (e.g., by phosphorylation or acetylation) proteins in milligram amounts in hours. Understanding protein function is a cornerstone of modern biology, and the same-day protein production described here is uniquely suited to accelerate these efforts.

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Section: Synthetic Side Products Misfoldsupporting

confidence: 80%

Section: Folding Selections Afford Purified Synthetic Proteinssupporting

confidence: 79%

Section: Synthetic Side Products Misfoldmentioning

confidence: 99%

Section: Diversification Of Folding Selection Protocols Affords a Var...mentioning

confidence: 99%

See 2 more Smart Citations

Same Day Access to Proteins Enabled by Folding Selections

Callahan

Rondon

Salazar

et al. 2023

Preprint

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show abstract

“…In the past two decades, many approaches have been explored to improve the efficiency of chemical protein synthesis, [2][3][4][5][6] but they all share inherent limitations in practice. One interesting new approach is automated flow chemistry, which has achieved initial success in synthesis of small proteins [7,8] ; it remains to be seen if it will fulfill its promise in wider application.…”

Section: Introductionmentioning

confidence: 99%

Development of convergent hybrid phase ligation for efficient and convenient total synthesis of proteins

Hong,

Yu,

Zhang

et al. 2023

Peptide Science

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Simple and efficient total synthesis of homogeneous and chemically modified protein samples remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP-NCL) strategy for facile preparation of proteins. In this strategy, proteins are split into $100-residue blocks, and each block is assembled on solid support from synthetically accessible peptide fragments before ligated together into full-length protein in solution. With the new method, we increase the yield of CENP-A synthesis by 2.5-fold compared to the previous hybrid phase ligation approach. We further extend the new strategy to the total chemical synthesis of 212-residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight peptide segments with only one single purification. We demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.

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Backbone‐Installed Split Intein‐Assisted Ligation for the Chemical Synthesis of Mirror‐Image Proteins

et al. 2023

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Membrane‐associated D‐proteins are an important class of synthetic molecules needed for D‐peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone‐Installed Split Intein‐Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L‐forms of the N‐ and C‐intein fragments of the unique consensus‐fast (Cfa) (i.e. L–CfaN and L–CfaC) are separately installed onto the two D‐peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (μM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D‐proteins without leaving any “ligation scar” on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D‐enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D‐protein targets.

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Single-Shot Flow Synthesis of D-Proteins for Mirror-Image Phage Display

Cited by 11 publications

References 52 publications

Same Day Access to Proteins Enabled by Folding Selections

Same Day Access to Proteins Enabled by Folding Selections

Development of convergent hybrid phase ligation for efficient and convenient total synthesis of proteins

Backbone‐Installed Split Intein‐Assisted Ligation for the Chemical Synthesis of Mirror‐Image Proteins

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