Protein and Peptide Letters - Current Issue

Tissue Factor (TF) is a crucial transmembrane glycoprotein that triggers blood coagulation upon vascular or tissue injury by binding to plasma factors VII and VIIa. In recent years, the demand for TF has rapidly increased due to its pivotal role in preoperative coagulation tests. However, large-scale production of TF remains challenging despite successful recombinant expression, as incorrect post-translational modifications adversely affect TF activity.

This study aims to investigate the role of post-translational modifications, specifically N-glycosylation, in TF activity and stability. Additionally, it explores strategies to enhance TF production by reducing its degradation through genetic modulation.

We compared TF activity derived from human cells and E. coli to assess the impact of post-translational modifications. Furthermore, we examined the effect of N-glycosylation on TF function. To address TF degradation, we knocked out the HRD1 gene, a key component of the endoplasmic-reticulum-associated degradation (ERAD) pathway, and evaluated its impact on TF stability and activity.

TF produced in human cells exhibited higher activity than TF expressed in E. coli, emphasizing the importance of post-translational modifications. Specifically, N-glycosylation was found to influence TF activity and stability. Additionally, we observed that knocking out the HRD1 gene effectively reduced TF degradation without compromising its activity.

Our findings underscore the crucial role of N-glycosylation in TF function and stability. Moreover, the modulation of the ERAD pathway through knocking out HRD1 presents a promising approach for enhancing TF production. These insights could contribute to the large-scale manufacturing of functionally active TF for clinical and research applications.

Angora goats are a distinct breed that differs significantly from common goats and shares a similar appearance to sheep. In Angora goats, only the level of glutathione (GSH) is elevated during under-stimulated conditions, as well as after the period of hypoxic stress; however, no changes are found in 2,3-diphosphoglycerate (2,3-DPG) levels, which are commonly present in the red blood cells (RBCs) of most mammals. We chose the Angora goat for our investigation because no previous studies have been conducted on the structural and functional aspects of hemoglobin (Hb). In addition, no sequence or structural information is currently available in any database.

Angora goat Hb was isolated and purified by anion-exchange chromatography, followed by crystallization using various methods. X-ray data collection for Angora goat Hb was performed under a liquid nitrogen cryo-stream using a Bruker D8 Venture Bio Photon III 28-pixel array area detector system.

Good diffracting crystals were obtained using the hanging-drop vapor-diffusion method with polyethylene glycol (PEG) 3350 as the precipitant in water, without the addition of any salt or buffer. The Angora goat Hb diffracted to a resolution of 1.85 Å, and the structure solution was obtained by the molecular replacement method, using the structure of domestic goat Hb as the starting model.

The solved structure of Angora goat crystallized in the monoclinic space group P21, consisting of one whole biological molecule in the asymmetric unit, with unit cell dimensions of a = 52.08 Å, b = 76.70 Å, c = 74.08 Å, and β = 91.77 °. The solvent content and Matthews coefficient (Vm) for the Angora goat Hb are 49.05% and 2.41 ų/Da, respectively, and are within the normal range for protein crystals.

Purification, crystallization, and preliminary X-ray diffraction studies of Angora goat Hb were performed successfully. Structural refinement and biophysical characterization of Angora goat Hb are in progress in the absence and presence of GSH and 2,3-DPG.

Keratinases have an established role in degrading highly stable and insoluble fibers of keratin proteins, which are otherwise difficult to be hydrolyzed by conventional proteases. Keratinases find promising application in degrading poultry waste to valuable products. Moreover, their role in cosmetics, detergents, agriculture and the leather industry is well recognized.

In this study, the keratinase gene from locally isolated Brevibacillus agri bacteria was cloned and expressed in Escherichia coli, and some of its potential applications were explored.

1300 bp amplified gene from Brevibacillus agri was cloned into E. coli DH5α competent cells using pTZ57R/T vector. After blue-white screening, the positive clone was confirmed by colony PCR and restriction analysis. Purified keratinase gene KerH from recombinant pTZR/KerH plasmid was ligated into pET-28a (+) and transferred into competent cells of E. coli DH5α. Following confirmation through colony PCR, and restriction analysis, recombinant plasmid (pET-28a/Ker) from the positive clone was transferred into competent E. coli BL21 cells. The transformed cells were then cultured for up to 8 hours after induction with 0.8 mM IPTG and lysed by sonication. The resulting recombinant keratinase (KerH) was purified by heat treatment and Ni-affinity column and characterized.

The blast analysis and homologous sequences in the NCBI database established a close link to Brevibacillus agri. The highest expression from transformed E. coli BL21 was achieved with 0.8 mM IPTG following 6 hours of induction. The resulting recombinant keratinase (KerH), purified by Ni-affinity chromatography, possessed 283 U/mg specific activity and displayed ~45 kDa band on SDS-PAGE and zymogram. Secondary structure analysis and active site prediction was performed computationally. Considering the extensive applications of keratinase, KerH was found to be useful in dehairing animal skin surfaces without any damage. The encapsulated KerH possessed improved stability and better compatibility with commercial detergents. It efficiently removed blood, turmeric, strawberry, and egg yolk stains from the fabric. Furthermore, KerH significantly degraded the poultry feathers and provided a protein hydrolysate that helped in converting damaged, dull and curly hair into healthier, shiny and straightened hair.

These key findings highlight that KerH is a robust keratinase with significant potential as an environmental-friendly alternative to the prevailing harsh chemical treatments in various industries. Encapsulation enhanced its suitability by improving its stability and shelf-life. Its broader substrate specificity, stability and application in detergents and cosmetics underline its commercial importance.

The recombinant KerH from Brevibacillus agri can be considered as a valuable microbial keratinase that can be used as an alternative to the eco hazardous chemicals used in commercial applications of feather degradation, hair protein treatment, feather keratin hydrolysate production and hide dehairing.