Preliminary Keratin Protein Purification through Liquid Chromatography Megan Forst, Victoria Khaimov, Dr. Roche C. de Guzman Fred DeMatteis School of Engineering and Applied Science, Hofstra University, Hempstead, NY 11549 INTRODUCTION Keratins are cytoskeletal structural proteins which are key structural components in hair, nails, and the outer layer of skin in vertebrates. Keratins assemble as type I and type II intermediate filament (IF) heterodimers. In hair, keratins are surrounded by keratin associated proteins (KAPs) which are tightly bound together (Fig1). Due to the covalent and noncovalent interactions within the hair strand, keratin proteins are commonly considered an insoluble material. In order to better understand the properties of keratins in-depth for efficient use in biomaterial research and other applications, keratins must be purified and isolated. An effective protein separation technique is liquid chromatography (Fig 2). Liquid chromatography utilizes interactions of the sample in both mobile and stationary phases in order to separate and identify components of a mixture.
Figure 1. Hair diagram showing the locations of keratin IFs and KAPs
METHODS Using a variety of solvents and additives, solubilities of KTN powder were obtained (Fig 3). Dissolved KTN samples in different basic pH buffers plus nonionic detergents and dithiothreitol (DTT) reducing agent were run using the AKTA Pure 25 L (Cytiva, formerly GE Healthcare) liquid chromatography system with AIEX HiTrap Q HP Column under varying conditions. Fractions collected were then characterized through spectrophotometry and gel electrophoresis.
Anion exchange chromatography did not effectively purify the keratin proteins. Therefore, further improvements to AIEX methods are necessary. Additionally, purification using other liquid chromatography techniques such as hydrophobic interaction (HIC) and sizeexclusion (SEC) will be explored in order to further isolate keratin proteins for a more purified result.
REFERENCES Figure 3. (Left) Crude KTN extracted from barbershop hair. (Right) Fully dissolved KTN solution in buffer at pH 7.5 with 5 M urea, 50 mM Tris, 10% glycerol, and 5 mM dithiothreitol (DTT).
Figure 4. Diagram of AIEX LC methodology during sample elution.
RESULTS OBJECTIVES ยง To determine the reduced crude hair extract (KTN) solubilities using a variety of reagents ยง To develop a purification method to separate keratin proteins from KTN using anion exchange (AIEX) liquid chromatography
Figure 5. Absorbance and conductivity graphs for protein and salt detection, respectively, which show successful separation of keratin fraction (confirmed via SDS-PAGE) from crude KTN. Figure 2. AKTA Pure 25 L liquid chromatography system
CONCLUSIONS
At pH 7.5, protein peaks were eluted using a linear gradient of 1 M NaCl. Two protein peaks were observed at approximately 660 mM NaCl and 1 M NaCl. Gel electrophoresis of fractions containing these peaks showed the existence of monomers (40-60 kDa), dimers (80-120 kDa), and multimers, consistent with the expected molecular weight of keratin; however, lower molecular weight signals were also evident indicating contamination with KAPs.
1.de Guzman RC, Tsuda SM, Ton MN, Zhang X, Esker AR, Van Dyke ME. Binding interactions of keratin-based hair fiber extract to gold, keratin, and BMP-2. PLOS ONE (2015) 10:e0137233. PMID: 26317522 2. Generical Electric Company. ร KTA Pure User Manual, Sweden (2012). 3. Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol (2008). 4. Plowman JE, Harland DP. Fibre Ultrastructure. Springer Nature Singapore Pte Ltd (2015)
ACKNOWLEDGMENTS This work was made possible through the equipment support of Hofstra University, Provost Office. KTN samples were provided by Dr. Roche de Guzman from his previous work at Virginia Tech.