Purification, crystallization and preliminary crystallographic analysis of mare lactoferrin.
Abstract: Lactoferrin is an iron-binding glycoprotein with a molecular weight of 80 kDa. The protein has two iron binding sites. It has two structural lobes, each housing one Fe(3+) and the synergistic CO(3)(2-) ion. The protein was isolated from the colostrum/milk of mares maintained at National Research Centre on Equines, Hisar, India. The purified samples of the protein were crystallized using a microdialysis method. The protein was dialysed against low ionic strength buffer solution. Several crystal forms were obtained, out of which three were characterized which have cell dimensions as follows. Form I a = 79.8, b = 103.5, c = 112.0 A, space group P2(1)2(1)2(1), with one protein molecule per asymmetric unit and a solvent content of 57%. Form II a = 84.9, b = 99.7, c = 103.5 A, space group P2(1)2(1)2(1) with one molecule per asymmetric unit and a solvent content of 55%. Form III a = 151.0, b = 151.0, c = 240.6 A, space group P4(1)2(1)2 with three molecules in the asymmetric unit and a solvent content of 57%. The intensity data up to 3.8 A resolution for form I, 2.9 A resolution data for form II and 6 A resolution data for form III have been collected. Further calculations are in progress.
Publication Date: 1996-11-01 PubMed ID: 15299585DOI: 10.1107/S0907444996007986Google Scholar: Lookup
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Summary
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The research describes the isolation, purification and subsequent crystallization of mare (horse) lactoferrin – an iron-binding protein, with preliminary exploration of its crystallographic structure.
Protein Isolation and Purification
- The research team focused on lactoferrin, a glycoprotein with a molecular weight of 80 kDa. This protein binds iron and has two iron binding sites.
- This protein was collected from the colostrum/milk of mares kept at the National Research Centre on Equines, based in Hisar, India.
- The isolated lactoferrin was purified for further analysis.
Protein Crystallization
- The purified protein samples were subjected to the process of crystallization using a microdialysis method, which uses dialysis to manipulate the concentration of solute in the sample. This method helped in forming protein crystals for further study.
- The protein sample was dialyzed against a buffer solution with low ionic strength to aid in crystal formation.
- Several crystal forms of the protein were obtained from this process.
Crystallographic Analysis
- Three distinct crystal forms among the obtained ones were identified and analyzed.
- Each form differed in terms of its dimensions, space group (symmetry), the number of protein molecules within each asymmetrical unit, and solvent content.
- These three forms were designated as Form I, II, and III in the study.
- Data on the intensity of reflections from the crystals were collected up to resolutions of 3.8 A, 2.9 A, and 6 A for Form I, II, and III respectively. Higher resolution means more details could be observed in the crystal structure.
- The authors highlight that further calculations based on these collected data are ongoing.
Conclusion
- The study provides preliminary analysis of the crystallographic properties of mare lactoferrin.
- These initial findings pave the way for further investigation into the specific structures and functions of the lactoferrin protein, which could have significant implications for understanding lactoferrin’s role in immune response and potentially its applications in clinical settings.
Cite This Article
APA
Sharma AK, Kathikeyan S, Kaur P, Singh TP, Yadav MP.
(1996).
Purification, crystallization and preliminary crystallographic analysis of mare lactoferrin.
Acta Crystallogr D Biol Crystallogr, 52(Pt 6), 1196-1198.
https://doi.org/10.1107/S0907444996007986 Publication
Researcher Affiliations
- Department of Biophysics, All India Institute of Medical Sciences, New Delhi.
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