Efficient isolation of the subunits of recombinant and pituitary glycoprotein hormones

• Published in: Journal of Chromatography A Vol. 1216; no. 9; pp. 1431 - 1438
• Main Authors: Carvalho, C.M., Oliveira, J.E., Almeida, B.E., Ueda, E.K.M., Torjesen, P.A., Bartolini, P., Ribela, M.T.C.P.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 27-02-2009; Amsterdam; New York: Elsevier; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; CHO Cells; Chromatography, High Pressure Liquid - methods; Cricetinae; Cricetulus; Fundamental and applied biological sciences. Psychology; Glycoprotein Hormones, alpha Subunit - isolation & purification; Glycoprotein Hormones, alpha Subunit - metabolism; hFSH; hLH; hTSH; Humans; Hydrophobic and Hydrophilic Interactions; Isoelectric Focusing; MALDI-TOF-MS; Pituitary Hormones, Anterior - isolation & purification; Pituitary Hormones, Anterior - metabolism; Protein hormones. Growth factors. Cytokines; Protein Subunits - isolation & purification; Protein Subunits - metabolism; Proteins; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RP-HPLC; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; α- and β-subunits; RP-HPLC; hTSH; MALDI-TOF-MS; hFSH; hLH; Isoelectric focusing; α- and β-subunits; HPLC chromatography; Thyroid stimulating hormone; Matrix assisted laser desorption ionization; Glycoprotein hormone; Subunit; Separation method; Luteinizing hormone; Recombinant protein; Follicle stimulating hormone; Human; Purification; Gel permeation chromatography; Reversed phase chromatography; Adenohypophyseal hormone; Time of flight method; Isolation; Mass spectrometry; Comparative study
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2008.12.096

Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37°C in acetic acid. The α-and β-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of >98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C4 column (80–90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.