Recombinant Human Keratin, type I cytoskeletal 27 (KRT27)

What is the structural classification of KRT27?

KRT27 is classified as a keratin belonging to the Type I family of intermediate filament proteins. In humans, it is encoded by a gene located on chromosome 17 . As a cytoskeletal protein, it contributes to cellular structure and function through its interaction with other keratins. Type I keratins are characterized by their acidic properties and typically form heterodimers with Type II keratins to create intermediate filaments that are essential for maintaining cellular integrity.

What are the primary challenges in producing recombinant KRT27?

Recombinant production of KRT27 faces similar challenges to other keratins. Traditional keratin extraction methods result in: (a) difficulty controlling amino acid composition; (b) batch-to-batch inconsistent quality; and (c) production of complex mixtures of keratin and keratin-associated proteins (KAPs) . These challenges have hindered mechanistic studies of keratin functions. Recombinant expression systems offer superior control over protein structure and purity but require optimization of expression conditions to ensure proper folding and solubility of the keratin proteins.

How does the secondary structure of recombinant KRT27 compare to naturally extracted keratins?

While specific data for KRT27 is limited in the search results, research on related keratins provides insight. Circular dichroism (CD) spectra analysis of recombinant keratins K37 and K81 demonstrated that these proteins are rich in α-helical secondary structures . This structural characteristic is likely shared by KRT27 as it belongs to the same keratin family. The α-helical structure is critical for the formation of coiled-coil dimers, which are the building blocks of keratin intermediate filaments.

What expression systems are most effective for producing recombinant KRT27?

Based on research with similar keratin proteins, heterologous expression systems such as E. coli or yeast have been successfully employed. For instance, type-I human hair keratin K37 and type-II human hair keratin K81 were heterologously expressed with significantly higher purity compared to extracted keratins . When choosing an expression system for KRT27, researchers should consider codon optimization, inclusion of appropriate tags for purification, and conditions that promote proper folding of the protein.

How do mutations in KRT27 affect hair morphology in different species?

Research has demonstrated that specific mutations in KRT27 significantly impact hair phenotypes across species. In cattle, the rs384881761 mutation in KRT27 results in a curly hair phenotype . KRT27 plays a pivotal role in determining hair morphology through keratin secretion, which ultimately shapes the final appearance of the hair . Understanding the molecular mechanisms by which these mutations alter keratin assembly and hair structure could provide insights into human hair disorders and potential therapeutic approaches.

What is the relationship between KRT27 expression and hair follicle cycle regulation?

KRT27 exhibits a specific spatial and temporal expression pattern in wool hair follicles, suggesting it plays an important regulatory role in hair follicle cycle regulation, particularly during the growth and degeneration phases . Recent research indicates that KRT27 acts as a factor that promotes hair growth, but its expression is inhibited by elevated homocysteine levels, potentially contributing to hair loss mechanisms . This relationship illustrates the complex regulatory networks governing hair follicle development and cycling.

How does RNA editing affect KRT27 function in normal versus pathological conditions?

RNA editing, particularly adenosine-to-inosine editing mediated by ADAR enzymes, may alter KRT27 expression and function. In keratoconus, a corneal disorder, abnormal RNA editing patterns have been observed in keratin gene clusters. While KRT27 was not specifically mentioned, related keratins showed differential editing between diseased and control samples . This suggests that post-transcriptional modifications could play a role in regulating KRT27 function and may contribute to pathological conditions affecting keratin-rich tissues.

What are the key genetic polymorphisms in KRT27 that correlate with hair quality traits?

A SNP at locus 1919G/A within KRT27 has been identified and studied in relation to cashmere goats . This polymorphism shows correlations with cashmere fiber characteristics, suggesting genetic variations in KRT27 contribute to differences in hair quality traits. The genotype frequency analysis revealed that the GA genotype at the 1919G/A locus was present at higher frequency in certain populations, indicating potential selective pressure on this genetic variant in relation to commercially valuable hair traits.

What purification techniques yield the highest purity recombinant KRT27?

For optimal purification of recombinant KRT27, researchers should implement a multi-step approach:

• Initial capture using affinity chromatography (if a tag system is employed)

• Intermediate purification with ion-exchange chromatography (leveraging KRT27's acidic properties)

• Final polishing with size-exclusion chromatography

SDS-PAGE analysis should be employed to confirm purity, as demonstrated with other recombinant keratins which showed significantly higher purity compared to extracted keratins . Western blotting with specific antibodies can confirm identity and integrity of the purified protein.

How can researchers effectively characterize the secondary structure of recombinant KRT27?

Secondary structure characterization of recombinant KRT27 should employ multiple complementary techniques:

• Circular Dichroism (CD) spectroscopy: This technique has been successfully used with recombinant keratins K37 and K81, revealing rich α-helical content

• Fourier-Transform Infrared Spectroscopy (FTIR): Provides additional structural information

• Limited proteolysis combined with mass spectrometry: Reveals accessible regions and domain organization

• X-ray crystallography or cryo-EM: For higher-resolution structural analysis, though these may be challenging due to keratin's propensity to form filaments

What are the most reliable methods for analyzing KRT27 gene polymorphisms?

The following methodological pipeline is recommended for KRT27 polymorphism analysis:

• PCR amplification using specific primers targeting the KRT27 gene region (primers should be designed based on conserved regions)

• DNA sequencing of the amplified fragments

• Analysis with software such as Chromas 2 and DNAMAN to identify SNPs (as demonstrated in the identification of the 1919G/A locus in KRT27)

• Calculation of genetic diversity parameters including:

  • Genotype and allele frequencies

  • Polymorphism information content (PIC)

  • Effective number of alleles (Ne)

  • Heterozygosity (He)

• Statistical analysis to correlate polymorphisms with phenotypic traits

What experimental designs are optimal for evaluating KRT27's role in tissue engineering applications?

Based on research with related keratins, a comprehensive experimental design should include:

• In vitro assessment:

  • Biocompatibility testing with relevant cell lines

  • Mechanical property characterization of KRT27-based scaffolds

  • Degradation kinetics under physiological conditions

• Functional testing:

  • Hemostatic potential evaluation through:

    • Fibrin clot formation assays

    • Blood coagulation time measurements

    • Platelet aggregation and adhesion studies

• In vivo models:

  • Liver puncture and femoral artery injury models (as used with K37 and K81)

  • Measurement of bleeding time and blood loss

  • Histological assessment of wound healing

  • Long-term biocompatibility evaluation

How does the hemostatic efficacy of recombinant KRT27 compare to other hemostatic agents?

While specific data on KRT27's hemostatic properties are not directly provided in the search results, research on related recombinant keratins (K37 and K81) demonstrated their ability to enhance fibrin clot formation at injury sites and decrease bleeding time and blood loss in liver puncture and femoral artery injury rat models . Researchers investigating KRT27's hemostatic potential should conduct comparative studies against both other recombinant keratins and conventional hemostatic agents, assessing parameters such as clotting time, clot strength, and effectiveness under various bleeding scenarios.

What are the potential applications of KRT27 in hair follicle development research?

KRT27's specific expression pattern in hair follicles and its role in hair morphology determination make it valuable for hair follicle development research . Researchers can utilize KRT27 as a marker for specific stages of follicle development or manipulate its expression to study effects on hair growth and structure. Additionally, the relationship between KRT27 expression and homocysteine levels suggests potential avenues for investigating biochemical regulators of hair growth and loss mechanisms.

How can transcriptomic analysis be leveraged to understand KRT27's role in different tissues?

Transcriptomic approaches such as RNA-seq can provide valuable insights into KRT27's expression patterns and regulation across tissues. Researchers should consider:

• Comparative transcriptomics of normal vs. pathological tissues

• Analysis of KRT27 co-expression networks to identify functional partners

• Investigation of RNA editing patterns affecting KRT27 expression or function

• Temporal analysis during development or tissue regeneration

RNA editing analysis, as demonstrated in keratoconus research, can reveal post-transcriptional regulation mechanisms that might affect KRT27 function .