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Structural requirements for the biosynthesis of ..

(2002) Heterologous expression and purification of SpaB involved in subtilin biosynthesis.

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to biosynthesis of backbone cyclic peptide ..

The increased proteolytic stability and enhanced binding affinity of conformationally constrained peptides make them attractive biosynthetic targets (, ). Toward this end, a method to generate libraries of cyclic peptides that employs circularly permuted inteins which self-catalyze the cyclization of a target peptide has been developed in Escherichia coli (–). This method, termed split intein catalyzed ligation of proteins and peptides (SICLOPPS), has been previously used to select inhibitors of hydrolases, methyl transferases, and other proteins (, , –). However, one major disadvantage of this system relative to nonribosomal peptide synthesis is the limited repertoire of functional groups that can be accessed. For example, chemical warheads such as ketoamides, boronates, hydroxamates, and Michael acceptors (which bind covalently to active site residues in proteases, acyl transferases, kinases, etc.), or N-methyl and β-amino acids (which can be used to block proteolysis or restrict conformation) are absent from the canonical 20 amino acids. Toward this end, we report an approach of a template-directed biosynthesis of large libraries of cyclic peptides containing UAAs in bacteria and the use of this library to evolve inhibitors of HIV protease in E. coli.

Accuracy in protein biosynthesis is maintained through multiple pathways, ..

(1999) Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD).

In a SICLOPPS construct, the ..

06-07-2017 · Combinatorial biosynthesis of cyclic lipopeptide antibiotics: a model for synthetic biology to accelerate the evolution of secondary metabolite biosynthetic pathways.

Recent advances in chemical biology and the advantages presented by in vivo screening have highlighted the need for a robust and flexible biologically synthesized small-molecule library. Herein we describe a method for the biosynthesis of cyclic peptide libraries of up to 108 members in Escherichia coli using split-intein circular ligation of peptides and proteins (SICLOPPS). The method utilizes split-intein chemistry to cyclize randomized peptide sequences. The cyclic peptide library can potentially be of any size and the peptide itself may contain unlimited random residues. However, the library size is limited by the transformation efficiency of E. coli and random residues are generally limited to five, but additional amino acids can be used in the cyclic peptide backbone, varying the structure and ring size of the cyclic peptide. SICLOPPS libraries have been combined with a bacterial reverse two-hybrid system in our labs and used in the identification of inhibitors of several protein–protein interactions. This protocol is expected to take around 3–4 weeks to implement.

Survivingcolonies from these plates were grown overnight and assessed by drop-spotting10-fold serial dilutions onto minimal media plates, supplementedwith antibiotics, IPTG and 3-AT as above, with and without 6.5 μM -arabinose. Plasmids from strains showing a growth advantagein the presence of arabinose were isolated and retransformed intothe original selection strain and reassessed for IPTG-dependent inhibitionof growth, and arabinose growth rescue. SICLOPPS plasmids from coloniesdemonstrating the expected phenotypes were assessed for their HIF-1specificity by transformation into two identical RTHS, except forthe replacement of HIF-1 with unrelated proteins (ATIC, a homodimericenzyme used in purine biosynthesis, and P6/UEV, a heterodimeric interactionrequired for the budding of HIV from infected cells)., Plasmids that caused a growth-advantage in the ATIC or P6/UEV RTHSwere discarded for being nonspecific. The activity of the cyclic peptidesencoded by the remaining SICLOPPS plasmids was ranked by retransforminginto the HIF-1 RTHS and drop spotting of 10-fold dilutions. The identityof the variable insert regions encoding the active cyclic peptideswas revealed by DNA sequencing.

for the biosynthesis of backbone cyclic peptide ..

to then move to the USA to pursue a PhD at Brown University, centred on studying the biosynthesis of polyketide …

21-08-2015 · As such, it provides a new approach, complementary in scope to those previously reported, 49, 50 to direct the biosynthesis of cyclic peptides of completely arbitrary sequence. Compared to SICLOPPS, for example, the present method offers the versatility to access the desired peptide macrocycle in a broader range of molecular …
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  • amino acid into SICLOPPS cyclic peptide libraries ..

    purine biosynthesis with ..

  • Biological Synthesis of Circular Polypeptides

    Department of Chemistry, ..

  • Volume 83, 2014 | Annual Review of Biochemistry

    Here we describe an intein-based approach to biosynthesis of backbone cyclic peptide ..

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