Cysteine‐Rich Peptides: Hyperstable Scaffolds for Protein Engineering

Abstract

Cysteine‐rich peptides (CRPs) are small proteins of less than 100 amino acids in length characterized by the presence of disulfide bridges and common end‐to‐end macrocyclization. These properties confer hyperstability against high temperatures, salt concentration, serum presence, and protease degradation to CRPs. Moreover, their intercysteine domains (loops) are susceptible to residue hypervariability. CRPs have been successfully applied as stable scaffolds for molecular grafting, a protein engineering process in which cysteine‐rich structures provide higher thermodynamic and metabolic stability to an epitope and acquire new biological function(s). This review describes the successes and limitations of seven cysteine‐rich scaffolds, their bioactive epitopes, and the resulting grafted peptides.

Milking the Cow: Cattle-Derived Chimeric Ultralong CDR-H3 Antibodies and Their Engineered CDR-H3-Only Knobbody Counterparts Targeting Epidermal Growth Factor Receptor Elicit Potent NK Cell-Mediated Cytotoxicity

“…In addition, 38 codons within IGHD8-2 can be readily mutated to cysteine via only one nucleotide exchange by a diversification process involving activation-induced cytidine deaminase (12), resulting in an unprecedented structural diversity of the knob region mediated by different disulfide bond patterns (15). Intriguingly, also nature-derived cysteine-rich miniproteins have been engineered for a plethora of applications (16)(17)(18) culminating in FDA-approved therapeutic entities Ziconotide and Linaclotide (19,20). Due to their structural peculiarities, bovine ultralong CDR-H3 antibodies have been harnessed to target different antigenic components, with most of them being related to infectious diseases such as HIV (21)(22)(23).…”

On the Utility of Chemical Strategies to Improve Peptide Gut Stability

“…More general strategies for molecular peptide stabilization involve polymer conjugation , and the use of cyclization to engineer rigid structures, ,− both of which can prevent hydrolysis by hindering protease access to cleavable bonds. N-to-C-terminal backbone cyclization and side-chain stapling via disulfide bonds are also key structural motifs in several natural peptide scaffolds that are proposed as stable templates to engineer drug leads via grafting small epitopes into their framework. − High thermal, enzymatic, and/or serum stability has been indicated for natural and engineered versions of cyclotides, , θ-defensins, , sunflower trypsin inhibitor (SFTI-1), , conotoxins, , and chlorotoxin . However, little is known about the utility of these scaffolds and modification strategies to specifically improve gut stability.…”

Recent research progress of biologically active peptides

“…Especially in recent years, the research on bioactive peptides has gradually become active, and the prospects are becoming more and more promising. The current research and development directions of BAPs are roughly as follows: (1) The discovery of new active peptides, structural analysis, and activity research, and so forth 19 ; (2) Development of targeted enzymatic hydrolysis technology for protein preparation of active peptides, including high‐efficiency and specific enzyme‐producing strains breeding, research on the joint action mechanism of complex enzyme systems, improvement of enzymatic hydrolysis technology, and so forth 20 ; (3) Separation of functional peptides and development of analytical techniques, especially high‐sensitivity, simple, and easy target peptide activity analysis and detection systems and analytical techniques 21 ; (4) Research on functional biological evaluation of peptides 22 ; (5) Development of bioactive peptide medicines and functional foods 23 ; (6) Engineering research on low‐cost, high‐purity, and mass production of bioactive peptides 24 ; (7) Application research of bioengineering technology in the preparation of active peptides, such as protein engineering, 25 enzyme engineering, 26 genetic engineering, 27 cell culture technology, 28 and so forth.…”

“…(3) Separation of functional peptides and development of analytical techniques, especially high-sensitivity, simple, and easy target peptide activity analysis and detection systems and analytical techniques 21 ; (4) Research on functional biological evaluation of peptides 22 ; (5) Development of bioactive peptide medicines and functional foods 23 ; (6) Engineering research on low-cost, highpurity, and mass production of bioactive peptides 24 ; (7) Application research of bioengineering technology in the preparation of active peptides, such as protein engineering, 25 enzyme engineering, 26 genetic engineering, 27 cell culture technology, 28 and so forth.…”