Biomimetic design of fibril-forming non-immunogenic collagen like proteins for tissue engineering.

Collagen, a key component of extracellular matrix serves as a linchpin for maintaining structural integrity and functional resilience. Concerns over purity and immunogenicity of animal-derived collagens have spurred efforts to develop synthetic collagen-based biomaterials. Despite several collagen mimics, there remains limited exploration of non-immunogenic biomaterials with the capacity for effective self-assembly. To combat the lacuna, collagen like protein (CLP) variants were rationally designed and recombinantly expressed, incorporating human telopeptide sequences (CLP-N and CLP-NC) and bioactive binding sites (CLP-NB). Circular dichroism analyses of the variants confirmed the triple helical conformation, with variations in thermal stability and conformation attributed to the presence of telopeptides at one or both ends of CLP. The variants had propensity to form oligomers, setting the stage for fibrillogenesis. The CLP variants were biocompatible, hemocompatible and supported cell proliferation and migration, particularly CLP-NB with integrin-binding sites. Gene expression indicated a lack of significant upregulation of inflammatory markers, highlighting the non-immunogenic nature of these variants. Lyophilized CLP scaffolds maintained their triple-helical structure and offered favorable biomaterial characteristics. These results accentuate the potential of designed CLP variants in tissue engineering, regenerative medicine and industrial sectors, supporting the development of biocompatible scaffolds and implants for therapeutic and cosmetic purposes.

A Unique Temperature-Induced Reverse Supramolecular Chirality-Assisted Gel-to-Gel Transition.

Supramolecular hydrogels typically undergo a gel-to-sol transition with heat, as intermolecular interactions within the gel weaken. Although gel-to-gel transitions during heating are rare, they may occur due to minor rearrangements caused by thermal forces in the supramolecular self-assembled structure. Here, an unprecedented temperature-induced gel-to-gel transition assisted by supramolecular chiral inversion in a hydrogel system is presented. The transition results from a left-handed M-type helix to a right-handed P-type helix, attributed to the π-system-conjugated amino acid, l-Tyrosine (Fm- l-Tyr). Upon solvent dilution, Fm-l-Tyr induces translucent hydrogel formed by entangled fibers with a kinetically stable left-handed M-type supramolecular helix. At 70 °C, hydrogel transforms into an opaque gel with a reverse supramolecular chirality yielding a thermodynamically stable right-handed P-type helix. Supramolecular chiral inversion is substantiated by two chiroptical methods. This unique gel-to-gel transition, accompanied by chiral inversion, is anticipated to attract attention, especially for applications sensitive to chirality.

Development of masked silica tanning system for sustainable leather production.

Amid mounting pressure on the long-term recyclability of chromium in tanned leather and the associated environmental hazards, the quest for an alternative, cleaner tanning system has gained tremendous momentum. In this context, our study explores the remarkable potential of silicates as a versatile platform for skin/hide tanning, circumventing the inherent risks and ecological threats posed by chromium exposure and leaching. We present an alternative approach of using a silica-based tanning system, employing a Taguchi model, to optimize a masked silica (MaSil) tanning product/process for achieving effective collagen stabilization. Our results demonstrate the significant advancements made in hydrothermal denaturation temperature, reaching an impressive 79 °C through precise Taguchi parameters-5% SiO2, masked with 0.3 mole of citrate salt, and a tanning process fixation pH of 4.5. Notably, the mechanical strength analysis reveals compliance with the stringent upper leather recommendation standards, validating the practicality and quality of MaSil crust leather. Moreover, our research highlights the unprecedented environmental benefits of the first reported application of Taguchi's approach to the MaSil tanning system. The developed tanning system remarkably reduces total dissolved solids (TDS), biological oxygen demand (BOD), chemical oxygen demand (COD), and overall water load by 68.4%, 25.4%, 59.5%, and 33.7%, respectively, heralding a promising era of water and environmental sustainability in the leather sector. This study holds the potential to transform leather production, wherein the envisioned future on the use of the Taguchi model and optimized MaSil tanning system could find a place in shaping a cleaner, greener, and more sustainable leather industry.

A bio-inspired silkworm 3D cocoon-like hierarchical self-assembled structure from π-conjugated natural aromatic amino acids.

The formation of spontaneous 3D self-assembled hierarchical structures from 1D nanofibers is a significant breakthrough in materials science. Overcoming the major challenges associated with developing these 3D structures, such as uncontrolled self-assembly, complex procedures, and machinery, has been a formidable task. However, the current discovery reveals that simple π-system (fluorenyl)-functionalized natural aromatic amino acids, phenylalanine (Fmoc-F) and tyrosine (Fmoc-Y), can form bio-inspired 3D cocoon-like structures. These structures are composed of entangled 1D nanofibers created through supramolecular self-assembly using a straightforward one-step process of solvent casting. The self-assembly process relies on π-π stacking of the fluorenyl (π-system) moieties and intermolecular hydrogen bonding between urethane amide groups. The cocoon-like structures are versatile and independent of concentration, temperature, and humidity, making them suitable for various applications. This discovery has profound implications for materials science and the developed advanced biomaterials, such as Fmoc-F and Fmoc-Y, can serve as flexible foundational components for constructing 3D fiber-based structures.

Collagen Mimicry with a Short Collagen Model Peptide.

Mimicking triple helix and fibrillar network of collagen through collagen model peptide(CMP) with short GPO tripeptide repeats is a great challenge. Herein, a minimalistic CMP comprising only five GPO repeats [(GPO)5 ] is presented. This novel approach involves the fusion of ultrashort peptide with the synergetic power of π-system and ß-sheet formation to short CMP (GPO)5 . Accordingly, a hydrogel-forming, fluorenylmethoxycarbonyl (Fmoc)-functionalized ultrashort peptide (NFGAIL) is fused at the N-terminus and phenylalanine at the C-terminus of (GPO)5 (Fmoc-NFGAIL-(GPO)5 -F-COOH, FmP-5GPO). At room temperature, it forms a robust triple helix in aqueous buffer solution and has a relatively high melting point of 35 °C. The fluorenyl motif stabilizes the triple helix by aromatic π-π interactions as in its absence, triple helix is not formed. NFGAIL, which forms a ß-sheet, also aids in triple helix stabilization via intermolecular hydrogen bonding and hydrophobic interactions. FmP-5GPO forms highly entangled nanofibrils with a micrometer length, which have excellent cell viability. The achievement of stable triple helix and fibrils in such a short CMP(FmP-5GPO) sequence is a challenging feat, and its significance in CMP-based biomaterials is undeniable. The present strategy highlights the potential for developing new CMP sequences through intelligent tuning of fusion peptides and GPO repeats.

Structural Insights into the Amyloid Fibril Polymorphism Using an Isotope-Edited Vibrational Circular Dichroism Study at the Amino Acid Residue Level.

Polymorphism is common in both in vitro and in vivo amyloid fibrils formed by the same peptide/protein. However, the differences in their self-assembled structures at the amino acid level remain poorly understood. In this study, we utilized isotope-edited vibrational circular dichroism (VCD) on a well-known amyloidogenic peptide fragment (N22FGAIL27) of human islet amyloid polypeptide (IAPf) to investigate the structural polymorphism. Two individual isotope-labeled IAPf peptides were used, with a 13C label on the carbonyl group of phenylalanine (IAPf-F) and glycine (IAPf-G). We compared the amyloid-like nanofibril of IAPf induced by solvent casting (fibril B) with our previous report on the same IAPf peptide fibril but with a different fibril morphology (fibril A) formed in an aqueous buffer solution. Fibril B consisted of entangled, laterally fused amyloid-like nanofibrils with a relatively shorter diameter (15-50 nm) and longer length (several microns), while fibril A displayed nanofibrils with a higher diameter (30-60 nm) and shorter length (500 nm-2 µm). The isotope-edited VCD analysis indicated that fibrils B consisted of anti-parallel ß-sheet arrangements with glycine residues in the registry and phenylalanine residues out of the registry, which was significantly different from fibrils A, where a mixture of parallel ß-sheet and turn structure with the registry at phenylalanine and glycine residues was observed. The VCD analysis, therefore, suggests that polymorphism in amyloid-like fibrils can be attributed to the difference in the packing/arrangement of the individual ß-strands in the ß-sheet and the difference in the amino acid registry. Our findings provide insights into the structural aspects of fibril polymorphism related to various amyloid diseases and may aid in designing amyloid fibril inhibitors for therapeutic purposes.

Redirecting the JAK-STAT signal blocks the SARS-CoV-2 replication.

The distinct disease progression patterns of severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) indicate diverse host immune responses. SARS-CoV-2 severely impairs type I interferon (IFN) cell signaling, resulting in uncontrolled late-phase lung damage in patients. For better pharmacological properties, cytokine modifications may sometimes result in a loss of biological activity against the virus. Here, we employed the genetic code expansion and engineered IFN-ß, a phase II clinical cytokine with 3-amino tyrosine (IFN-ß-A) that reactivates STAT2 expression in virus-infected human cells through JAK/STAT cell signaling without affecting signal activation and serum half-life. This study identified that genetically encoded IFN-ß-A might stabilize the protein-receptor complex and trigger JAK-STAT cell signaling, which is a promising modality for controlling SARS-CoV-2 infection.

A Novel Surfactant with Short Hydrophobic Head and Long Hydrophilic Tail Generates Vesicles with Unique Structural Feature.

Surfactant molecules typically have a long hydrophobic tail and a short hydrophilic head group. It remains unexplored if surfactants can have a short hydrophobic head group and a long hydrophilic tail. Designing such surfactants is a challenge as a lengthy hydrophilic tail would completely solubilize the molecules. In this context, herein, the Fmoc-functionalized Gly-Pro-Hyp (GPO) tripeptide repeat-based molecule (Fm-GPO) with fluorenyl moiety as a short hydrophobic head and peptide as a long hydrophilic tail is demonstrated as a reverse surfactant at physiological pH, for the first time. π-π stacking of the fluorenyl moieties and intermolecular hydrogen bonding between the peptide chains with extended polyproline-II structure promoted the self-assembly into spherical vesicles with a unique feature of a large hydrophilic area in the interior and exterior of the bilayer. The current Fm-GPO system offers a new class of surfactants with unique features that can aid in the design of drug-loaded vehicles, which can be target-specific as the peptide chain can be manipulated with different functional ultra-short peptide sequences.

π-System Functionalization Transforms Amyloidogenic Peptide Fragment of Human Islet Amyloid Polypeptide into a Super Hydrogelator.

While a considerable number of ultra-short/short amyloid peptides have been reported to form 3D supramolecular hydrogels, they all possess high minimum gelation concentration (MGC) (≥1 wt%), which preclude their applications. In this context, we demonstrate that functionalisation of a well-known amyloidogenic ultra-short peptide fragment NFGAIL (IAPf) of human Islet amyloid polypeptide with a π-system (Fluorenyl, Fm) at the N-terminus of the peptide (Fm-IAPf) yield not only highly thermostable hydrogel at physiological pH but also exhibited super gelator nature as the MGC (0.08 wt%) falls below 0.1 wt%. Various experimental results confirmed that aromatic π-π interactions from fluorenyl moieties and hydrogen bonding interactions between the IAPf drive the self-assembly/fibril formation. Fm-IAPf is the first super hydrogelator derived from amyloid-based ultra-short peptides, to the best of our knowledge. We strongly believe that this report, i. e., functionalization of an amyloid peptide with π-system, provides a lead to develop super hydrogelators from other amyloid-forming peptide fragments for their potential applications.

Electrochemical Response of Redox Amino Acid Encoded Fluorescence Protein for Hydroxychloroquine Sensing.

The sudden rise in the demand has led to large-scale production of hydroxychloroquine (HCQ) in the global market for various diseases such as malaria, rheumatic arthritis, and systemic lupus erythematous and prophylactic treatment of early SARS-CoV-2 outbreak. Thorough monitoring of HCQ intake patients is in high demand; hence, we have developed a redox amino acid encoded fluorescent protein-based electrochemical biosensor for sensitive and selective detection of HCQ. This electrochemical biosensor is generated based on the two-electron transfer process between redox amino acid (3,4-dihydroxy-L-phenylalanine, DOPA) encoded bio-redox protein and the HCQ forms the conjugate. The DOPA residue in the bio-redox protein specifically binds with HCQ, thereby producing a remarkable electrochemical response on the glassy carbon electrode. Experimental results show that the developed biosensor selectively and sensitively detects the HCQ in spiked urine samples. The reagent-free bio-redox capacitor detects HCQ in the range of 90 nM to 4.4 µM in a solution with a detection limit of 58 nM, signal to noise ratio of 3:1, and strong anti-interference ability. Real-time screening, quantification, and relative mean recoveries of HCQ on spiked urine samples were monitored through electron shuttling using bio-redox protein and were found to be 97 to 101%. Overall, the developed bio-redox protein-based sensor has specificity, selectivity, reproducibility, and sensitivity making it potentially attractive for the sensing of HCQ and also applicable to clinical research.

Engineering microbial cells with metal chelating hydroxylated unnatural amino acids for removable of synthetic pollutants from water.

Lead (Pb2+) is a well-known heavy metal and toxic synthetic industrial pollutant in the ecosystem and causes severe threats to living organisms. It is paramount to develop a sustainable microbial engineering approach to remove synthetic pollutants from the environment. Genetic code engineering is emerging as an important microbial engineering tool in biosciences to biosynthesis congener protein production beyond the canonical set of natural molecules and expand the chemistries of living cells. Here, we prepare cells expressing unnatural amino acid encoded congener proteins for effectively removable toxic synthetic industrial pollutants (Pb2+) with high binding efficiency. Native and the developed congener proteins expressing cells adapted the Langmuir and Sips adsorption model that recommends uniform adsorption with Pb2+ ions. This could be due to a more significant number of functional groups on the protein surface. Fluorescence spectroscopic, field emission scanning electron microscope, X-ray photoelectron spectroscopic analysis, and protein-metal molecular stimulation coordination allowed us to explore the role of hydroxylation on Pb2+ adsorption. The bioreactor filled with immobilized protein-containing active granules showed >90% of lead removal in the contaminated water samples. The desorption of bound Pb2+ from GFP and its variants were studied by varying the pH to reuse the proteins for subsequent usage. We observed that about 70% of the GFP and its variants could be recycled and >75% of fluorescence efficiency could be recovered. Among all the variants, GFPHPDP exhibits high affinity and maintains the reusability efficiency in 7 consecutive cycles. These results suggest that genetic code engineering of cells encoding unnatural amino acids could be a next-generation microbial engineering tool for manipulating and developing the microbial strain's selective and effective removal of synthetic pollutants from the environment.

Cardiac surgery elicits pericardial inflammatory responses that are distinct compared with postcardiopulmonary bypass systemic inflammation.

Objectives: Cardiac surgery using cardiopulmonary bypass contributes to a robust systemic inflammatory process. Local intrapericardial postsurgical inflammation is believed to trigger important clinical implications, such as postoperative atrial fibrillation and postsurgical intrathoracic adhesions. Immune mediators in the pericardial space may underlie such complications. Methods: In this prospective pilot clinical study, 12 patients undergoing isolated coronary artery bypass graft surgery were enrolled. Native pericardial fluid and venous blood samples (baseline) were collected immediately after pericardiotomy. Postoperative pericardial fluid and venous blood samples were collected 48-hours after cardiopulmonary bypass and compared with baseline. Flow cytometry determined proportions of specific immune cells, whereas multiplex analysis probed for inflammatory mediators. Results: Neutrophils are the predominant cells in both the pericardial space and peripheral blood postoperatively. There are significantly more CD163lo macrophages in blood compared with pericardial effluent after surgery. Although there are significantly more CD163hi macrophages in native pericardial fluid compared with baseline blood, after surgery there are significantly fewer of these cells present in the pericardial space compared with blood. Postoperatively, concentration of interleukin receptor antagonist 6, and interleukin 8 were significantly higher in the pericardial space compared with blood. After surgery, compared with blood, the pericardial space has a significantly higher concentration of matrix metalloproteinase 3, matrix metalloproteinase 8, and matrix metalloproteinase 9. The same trend was observed with transformational growth factor ß. Conclusions: Cardiac surgery elicits an inflammatory response in the pericardial space, which differs from systemic inflammatory responses. Future work should determine whether or not this distinct local inflammatory response contributes to postsurgical complications and could be modified to influence clinical outcomes.

A "self-shrinking" supramolecular hydrogel with a 3D shape memory performance from an unnatural amino acid derivative.

A supramolecular hydrogel with 3D self-shrinking, without any assistance, and a shape memory performance at room temperature is discovered from an unnatural amino acid derivative, i.e. fluorenylmethoxycarbonyl-L-ß-phenylalanine, as a minimalistic model. The self-shrinking properties of this hydrogel can be explored for potential applications.

Isotope-edited vibrational circular dichroism study reveals a flexible N-terminal structure of islet amyloid peptide (NFGAIL) in amyloid fibril form: A site-specific local structural analysis.

The short peptide fragment NFGAIL (IAPf) is a well-known amyloidogenic peptide (22-27), derived from human islet amyloid polypeptide(hIAPP), whose fibrillar structure is often used to better understand the wild-type hIAPP amyloid fibrils, associated with type II diabetes. Despite an extensive study, the fibrillar structure of IAPf at the amino acid residue level is still unclear. Herein, the vibrational circular dichroism(VCD) spectroscopic technique coupled with isotope labelling strategy has been used to study the site-specific local structure of IAPf amyloid fibrils. Two 13C labeled IAPfs were designed and used along with unlabelled IAPf to achieve this. The 13C labelled (on -C=O) glycine(IAPf-G) and phenylalanine (IAPf-F) residues were introduced into the IAPf sequence separately by replacing natural glycine (residue 24) and phenylalanine (residue 23), respectively. VCD spectral analysis on IAPf-G suggests that IAPf fibrils adopt parallel ß-sheet conformation with glycine residues are part of ß-sheet and in-register. Unlike IAPf-G, VCD analysis on IAPf-F reveals that phenylalanine residues exist in the turn/hairpin conformation rather than ß-sheet region. Both VCD results thus suggest that IAPf amyloid fibril consists of a mixture of ß-sheet as a major conformation involving GAIL and turn/hairpin as a minor conformation involving NF rather than an idealized ß-sheet involving all the amino acids. While previous studies speculated that the full NFGAIL sequence could participate in the ß-sheet formation, the present site-specific structural analysis of IAPf amyloid fibrils at residue level using isotope-edited VCD has gained significant attention. Such residue level information has important implications for understanding the role of NFGAIL sequence in the amyloid fibrillation of hIAPP.

Recombinant and genetic code expanded collagen-like protein as a tailorable biomaterial.

Collagen occurs in nature with a dedicated triple helix structure and is the most preferred biomaterial in commercialized medical products. However, concerns on purity, disease transmission, and the reproducibility of animal derived collagen restrict its applications and warrants alternate recombinant sources. The expression of recombinant collagen in different prokaryotic and eukaryotic hosts has been reported with varying degrees of success, however, it is vital to elucidate the structural and biological characteristics of natural collagen. The recombinant production of biologically functional collagen is restricted by its high molecular weight and post-translational modification (PTM), especially the hydroxylation of proline to hydroxyproline. Hydroxyproline plays a key role in the structural stability and higher order self-assembly to form fibrillar matrices. Advancements in synthetic biology and recombinant technology are being explored for improving the yield and biomimicry of recombinant collagen. It emerges as reliable, sustainable source of collagen, promises tailorable properties and thereby custom-made protein biomaterials. Remarkably, the evolutionary existence of collagen-like proteins (CLPs) has been identified in single-cell organisms. Interestingly, CLPs exhibit remarkable ability to form stable triple helical structures similar to animal collagen and have gained increasing attention. Strategies to expand the genetic code of CLPs through the incorporation of unnatural amino acids promise the synthesis of highly tunable next-generation triple helical proteins required for the fabrication of smart biomaterials. The review outlines the importance of collagen, sources and diversification, and animal and recombinant collagen-based biomaterials and highlights the limitations of the existing collagen sources. The emphasis on genetic code expanded tailorable CLPs as the most sought alternate for the production of functional collagen and its advantages as translatable biomaterials has been highlighted.

Three-dimensional tailor-made collagen-like proteins hydrogel for tissue engineering applications.

Despite the potential tunable properties of blank slate collagen-like proteins (CLP), an alternative to animal-originated collagen, assembling them into a stable 3D hydrogel to mimic extracellular matrix is a challenge. To address this constraint, the CLP (without hydroxyproline, CLPpro) and its variants encoding functional unnatural amino acids such as hydroxyproline (CLPhyp) and 3,4-dihydroxyphenylalanine (CLPdopa) were generated through genetic code engineering for 3D hydrogel development. The CLPhyp and CLPdopa were chosen to enhance the intermolecular hydrogen bond interaction through additional hydroxyl moiety and thereby facilitate the self-assembly into a fibrillar network of the hydrogel. Hydrogelation was induced through genipin as a cross-linker, enabling intermolecular cross-linking to form a hydrogel. Spectroscopic and rheological analyses confirmed that CLPpro and its variants maintained native triple-helical structure, which is necessary for its function, and viscoelastic nature of the hydrogels, respectively. Unlike CLPpro, the varients (CLPhyp and CLPdopa) increased pore size formation in the hydrogel scaffold, facilitating 3T3 fibroblast cell interactions. DSC analysis indicated that the stability of the hydrogels got increased upon the genetic incorporation of hydroxyproline (CLPhyp) and dopa (CLPdopa) in CLPpro. In addition, CLPdopa hydrogel was found to be relatively stable against collagenase enzyme compared to CLPpro and CLPhyp. It is the first report on 3D biocompatible hydrogel preparation by tailoring CLP sequence with non-natural amino acids. These next-generation tunable CLP hydrogels open a new venue to design synthetic protein-based biocompatible 3D biomaterials for tissue engineering applications.

A cyclodextrin-based macrocyclic oligosaccharide cavitand with a dual functionality limits the collagen fibrillogenesis: A possible carbohydrate-based therapeutic molecule for fibrotic diseases.

ß-Cyclodextrin (ß-CD), a macrocyclic oligosaccharide cavitand, is a well-known candidate for drug delivery and formulation. In this study, we extended the application of ß-CD using a ß-cyclodextrin sulfate (ß-CDS) as a possible therapeutic for fibrotic diseases caused by excess deposition of collagen fibrils. We have strategically chosen ß-CDS, which mimics the natural existence of dermatan sulfate in the extracellular matrix, for limiting collagen fibrillation. The hydrophobic nature of the inner core ß-CDS is expected to form an inclusion complex with hydrophobic side chain amino acids with the simultaneous action of forming an ionic bond through a negative charge on sulfate group with positively charged amino acids side chain in collagen. Various results suggested that such dual action not only limited the collagen fibrillation but also reduced the fibril size formed in the presence of ß-CDS. The contemporary results thus indicate that ß-CDS can be explored as a therapeutic molecule in fibrotic diseases.

Comprehensive characterization of the postoperative pericardial inflammatory response: Potential implications for clinical outcomes.

Objective: There is a paucity of data on the inflammatory response that takes place in the pericardial space after cardiac surgery. This study provides a comprehensive assessment of the local postoperative inflammatory response. Methods: Forty-three patients underwent cardiotomy, where native pericardial fluid was aspirated and compared with postoperative pericardial effluent collected at 4, 24, and 48 hours' postcardiopulmonary bypass. Flow cytometry was used to define the levels and proportions of specific immune cells. Samples were also probed for concentrations of inflammatory cytokines, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Results: Preoperatively, the pericardial space mainly contains macrophages and T cells. However, the postsurgical pericardial space was populated predominately by neutrophils, which constituted almost 80% of immune cells present, and peaked at 24 hours. When surgical approaches were compared, minimally invasive surgery was associated with fewer neutrophils in the pericardial space at 4 hours' postsurgery. Analysis of the intrapericardial concentrations of inflammatory mediators showed interleukin-6, MMP-9, and TIMP-1 to be highest postsurgery. Over time, MMP-9 concentrations decreased significantly, whereas TIMP-1 levels increased, resulting in a significant reduction of the ratio of MMP:TIMP after surgery, suggesting that active inflammatory processes may influence extracellular matrix remodeling. Conclusions: These results show that cardiac surgery elicits profound alterations in the immune cell profile in the pericardial space. Defining the cellular and molecular mediators that drive pericardial-specific postoperative inflammatory processes may allow for targeted therapies to reduce immune-mediated complications.

Genetically encoded dihydroxyphenylalanine coupled with tyrosinase for strain promoted labeling.

Protein modifications through genetic code engineering have a remarkable impact on macromolecule engineering, protein translocation, protein-protein interaction, and cell biology. We used the newly developed molecular biology approach, genetic code engineering, for fine-tuning of proteins for biological availability. Here, we have introduced 3, 4-dihydroxy-l-phenylalanine in recombinant proteins by selective pressure incorporation method for protein-based cell labeling applications. The congener proteins treated with tyrosinase convert 3, 4-dihydroxy-l-phenylalanine to dopaquinone for strain-promoted click chemistry. Initially, the single-step Strain-Promoted Oxidation-Controlled Cyclooctyne-1,2-quinone Cycloaddition was studied using tyrosinase catalyzed congener protein and optimized the temporally controlled conjugation with (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol. Then, the feasibility of tyrosinase-treated congener annexin A5 with easily reactive quinone functional moiety was conjugated with fluorescent tag dibenzocyclooctyne-PEG4-TAMRA for labeling of apoptotic cells. Thus, the congener proteins-based products demonstrate selective cell labeling and apoptosis detection in EA.hy926 cells even after the protein modifications. Hence, genetic code engineering can be coupled with click chemistry to develop various protein-based fluorescent labels.