Chemical and semisynthesis of posttranslationally modified proteins.

Application of chemically synthesized proteins to the study of biochemistry and biophysics.2.
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Chemical synthesis and expression of the HIV‐1 rev protein.


By using the General Optimized Procedure, SEAoff peptide IRNC(StBu)IIGKGRSYKGTVSITKSGIK-SEAoff was converted into MPA thioester IRNCIIGKGRSYKGTVSITKSGIK-SCH2CH2CO2H with an isolated yield of 41% following HPLC purification (25 mg scale).[41] In another experiment, we isolated the same MPA thioester in 66 % yield by desalting the crude product on a C18 solid phase extraction column. Most of the time, a high resolution HPLC purification step is not required because i) the starting SEAoff peptide 3 is purified in the previous step and ii) the exchange reaction is very clean and proceeds in high yields. Note that Cys(StBu) residues are reduced into Cys during this step.

Convergent Chemical Synthesis of Proteins by Ligation of Peptide Hydrazides.
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Protein Chemical Synthesis by Ligation of Peptide Hydrazides


46. Banerjee SR, Maresca KP, Stephenson KA, Valliant JF, Babich JW, Graham WA, Barzana M, Dong Q, Fischman AJ, Zubieta J. N,N-bis(2-mercaptoethyl)methylamine: a new coligand for Tc-99m labeling of hydrazinonicotinamide peptides. Bioconjugate Chem. 2005; 16: 885-902.
47. Vojkovsky T. Detection of secondary amines on solid phase. Pept. Res. 1995; 8: 236-237.

Synthesis of Cyclic Peptides and Cyclic Proteins via Ligation of Peptide Hydrazides.
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Our initial efforts to fold these synthetic peptides using GroEL/ES resulted in measurable enzymatic activity, albeit at relatively low levels (∼20–40%; ), likely due to microheterogeneity in the synthetic peptides (). Because active DapA assembles as a tetramer, we reasoned that we could enrich for “foldable” protein by using a chemical refolding procedure followed by size-exclusion chromatography (SEC).

Recent extensions to native chemical ligation for the chemical synthesis of peptides and proteins.
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Convergent chemical synthesis of proteins by ligation …


29. Fang G-M, Li Y-M, Shen F, Huang Y-C, Li J-B, Lin Y, Cui H-K, Liu L. Protein chemical synthesis by ligation of peptide hydrazides. Angew. Chem. Int. Ed. 2011; 50: 7645-7649.

Protein Chemical Synthesis by Ligation of Peptide Hydrazides.


28. Tofteng AP, Sørensen KK, Conde-Frieboes KW, Hoeg-Jensen T, Jensen KJ. Fmoc solid-phase synthesis of C-terminal peptide thioesters by formation of a backbone pyroglutamyl imide moiety. Angew. Chem. Int. Ed. 2009; 48: 7411-7414.

Chemical synthesis of proteins using peptide ..

We reasoned that we could simplify the assembly if we eliminated the desulfurization step necessary to convert the Cys to native Ala at the DapA 2–3 junction. Toward this end, we determined locations in our protein that would likely tolerate permanent mutation to Cys. BLAST analysis of the E. coli DapA identified the 1,000 most-similar homologs (>69% conservation, >49% identity), which were aligned to determine positions where Cys residues naturally occur. Fortuitously, 12% of the aligned sequences contained Cys at position 77, site of the DapA 2–3 junction (). Next, we analyzed the DapA crystal structure to determine the likelihood of the A77C mutation to disrupt protein structure/function. The side chain of residue 77 is surface-exposed and not in close proximity to the active site or any native Cys residues (>12 Å to the nearest Cys; ). This analysis suggested that introduction of the A77C mutation would likely be well tolerated. Indeed, this mutation affected neither recombinant protein activity () nor its dependence on GroEL/ES for folding under physiological conditions ( and ). We ultimately selected a final assembly strategy that incorporated both the A77C mutation and a unified DapA 7–8 segment (we were not able to produce high-quality DapA 1–2, 3–4, or 5–6 unified peptides). This final strategy yielded a seven-segment assembly scheme () that removed four synthetic steps (and associated purifications) from the initial scheme.

Chemical synthesis of proteins using peptide hydrazides …


24. Sewing A, Hilvert D. Fmoc-compatible solid-phase peptide synthesis of long C-terminal peptide thioesters. Angew. Chem. Int. Ed. 2001; 40: 3395-3396.