Protein & Peptide Letters, 2007, 14, 87-91 87 0929-8665/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd. Large Scale Preparation of the Mammalian High Mobility Group Protein A2 for Biophysical Studies Tengjiao Cui 1 , Suzanne Joynt 1 , Victor Morillo 1 , Maria Baez 1 , Zhichun Hua 2 , Xiaotang Wang 1 and Fenfei Leng 1, * 1 Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8 th Street, Miami, FL 33199, USA and 2 The State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, P.R. China Abstract: Due to asymmetrical charge distribution of the mammalian high mobility group protein A2 (HMGA2), which makes HMGA2 bind to both cation- and anion-exchange columns, we developed a rapid procedure for purifying HMGA2 in the milligram range. This purification procedure greatly facilitated biophysical studies, which require large amounts of the protein. Keywords: The mammalian high mobility group protein A2 (HMGA2), DNA binding protein, isothermal titration calorimetry, differential scanning calorimetry, nuclear magnetic resonance (NMR) Spectroscopy, intrinsically unstructured protein. INTRODUCTION The mammalian high mobility group protein A2 (HMGA2) is a nuclear oncoprotein [1, 2]. Abnormal expres- sion of HMGA2 and its mutants is directly linked to tumori- genesis of a variety of benign tumors of mesenchymal origin such as lipomas, uterine leiomyomas, and fibroadenomas [3-6]. These solid tumors are very common in humans. For example, approximately 39% of all hysterectomies per- formed in the United States annually are uterine leiomyomas (1,361,786 cases from 1994 to 1999 [7]) and up to 77% of women of reproductive age may have these benign smooth-muscle tumors [8]. In many cases, rearrangements of chromosomal bands 12q13-15 where the HMGA2 gene is located cause disruptions of HMGA2’s normal functions [9, 10], which correlates with the formation of the tumors. Over-expression and deregulation of HMGA proteins in- cluding HMGA2 also causes several types of malignant tu- mors such as lung cancer [11], hepatocellular carcinoma [12], prostate cancer [13], oral cancer [14], and leukemia [15]. The expression level is correlated with the degrees of malignancy and metastatic potential of the transformed cells [9]. A recent study showed that HMGA proteins were ex- pressed in about 90% of lung carcinomas (152 cases), and the expression level is inversely associated with survival rate and prognosis [11]. These results suggest that HMGA pro- teins could be used as a biomarker for diagnosing the neo- plastic transformation and the metastatic potential of many cancers [16, 17]. HMGA2 is only expressed in proliferating, undifferenti- ated mesenchymal cells and is undetectable in normal fully differentiated adult cells [18, 19]. Disruption of its normal expression patterns causes deregulations of cell growth and *Address correspondence to this author at the Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8 th Street, Miami, Florida 33199, USA; Tel: 305-348-3277; Fax: 305-348-3772; E-mail: lengf@fiu.edu differentiation. Results from Chada’s laboratory showed that Hmga2 knock-out mice developed the pygmy phenotype [18]. These mutant mice were severely deficient in fat cells and other mesenchymal tissues (almost 20-fold decrease). Furthermore, it was discovered that disruption of the Hmga2 gene caused a dramatic reduction in obesity of leptin-deficient mice (Lep ob /Lep ob ) in a gene-dosage de- pendent manner: Hmga2 +/+ Lep ob /Lep ob mice weighed over three times more than Hmga2 -/- Lep ob /Lep ob animals, and the weight of Hmga2 +/- Lep ob /Lep ob mice was in-between [20]. These results suggest that HMGA2 plays an important role in fat cell proliferation and is a potential target for the treatment of obesity [20]. Due to its role in tumorigenesis and obesity described above, HMGA2 has been widely studied by a variety of bio- chemical and biophysical methods [21-23]. Unfortunately, it was difficult to purify large quantities of recombinant HMGA2 for biophysical studies such as nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) studies. One reason is that over-expression of HMGA2 is toxic to the host cells [24], which results in low level of ex- pression of the protein. Another reason is the lack of a rapid purification procedure for large scale preparation of HMGA2. As reported previously [25], one intriguing feature of HMGA2 is the asymmetrical charge distribution of its primary structure (Fig. 1). The positive charges are concen- trated in the three “AT hook” DNA binding domains, which tightly binds to the minor groove of AT-rich DNA sequences [23, 26]. The negative charges are mainly located in the C-terminus. This unique feature allowed for development of a rapid purification procedure to prepare HMGA2 in large quantities for various biophysical studies. Using a combina- tion of a strong cation-exchange column, such as SP-Sepharose FF column, and an anion-exchange column, such as Q-Sepharose FF column, we routinely purify 50 to 100 mg of HMGA2 from 10 liters of E. coli cell culture over-expressing recombinant HMGA2 (>98% pure).