Rosado Arroyo, Marie C.

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    Expression, isolation, purification, and characterization of recombinant human SFi1p1-2
    (2013) Rosado Arroyo, Marie C.; Pastrana Ríos, Belinda; College of Arts and Sciences - Sciences; Ríos, Robert; Ríos Steiner, Jorge; Department of Chemistry; Colón, Silvestre
    Homo sapiens Sfi1 (Hs Sfi1) is a 1242 amino acid protein containing 23 tandem binding sites for a calcium binding protein known as centrin. Hs Sfi1p₁₋₂ is an 8,592 Da peptide that corresponds to the first two centrin binding sites (CBS) within Hs Sfi1. Both proteins are constituents of contractile fibers in the centrosome and play essential roles in centriole duplication and separation. The objectives of this work were to: express, isolate, purify, and biochemically characterize recombinant Hs Sfi1p₁₋₂. A bacterial stock culture of Escherichia coli BL21-(DE3) RIL was transformed with pET 100 expression vector containing His tagged-Hs Sfi1p₁₋₂. His tagged-Hs Sfi1p₁₋₂ recombinant peptide has a molecular weight of 12,119 Da. The bacterial stock was grown in a five liter bench scale fermentor up to log phase and induced with isopropyl-β-thiogalactoside (IPTG). Following the successful expression of recombinant His-Hs Sfi1p1-2, the supernatant was subjected to His-tag affinity and anion exchange chromatography and a band was observed near the expected molecular weight of ~12 kDa by 4-20% (Bis-Tris) gradient SDS-PAGE. However, based on the elution pattern and UV/Vis analysis it was suspected that the recombinant peptide stayed in the pellet. An alternative isolation process was performed by an extraction method with organic solvents using CHCl₃:CH3OH (2:1, v/v) extraction followed by a CHCl₃:CH3OH (1:1, v/v) volume ratios. A 4-20% (Bis-Tris) gradient SDSPAGE revealed the presence of a protein with similar molecular weight in both the aqueous and the organic phases. Partial amino acid sequencing confirmed the presence of Hs Sfi1p1-2 to be in the aqueous phase of the extraction. Alternate purification step involved subjecting the protein sample to size exclusion chromatography.