Journal of Cell and Molecular Biology - ResearchGate

More documents

Recommendations

Info

Journal of Cell and Molecular Biology 9(2):37-42, 2011 Research Article 37 Haliç University, Printed in Turkey. http://jcmb.halic.edu.tr Cloning, expression, purification, and quantification of the 17% Nterminal domain of apolipoprotein b-100 Hassan M. KHACHFE * 1, 2, 3 and David ATKINSON 3 1 Faculty of Sciences-V, Lebanese University, Nabatieh, Lebanon 2 Departments of Biological and Biomedical Sciences, Lebanese International University, Beirut, Lebanon 3 Department of Physiology and Biophysics and Center For Advanced Biomedical Research, Boston University School of Medicine, 715 Albany Street, Boston MA 02118, USA (*author for correspondence; hassan.khachfe@liu.edu.lb ) Received: 22 August 2011; Accepted: 9 December 2011 Abstract Apolipoprotein B-100 (apo B) is the sole protein component of normal human low density lipoprotein (LDL). Elevated levels of LDL have been correlated with atherosclerosis and other coronary artery diseases. The large size of apo B (4536 aa) necessitates that it be studied in pieces corresponding to its structurally organized domains. The 17% N-terminal domain of apo B, simply B17, poses as one of these domains, having very specific structural characteristics. The current report describes a set of protocols for the cloning, expression, purification, and quantification of this important part of the protein. Keywords: Apolipoprotein (Apo B), C127 cells, cloning, low-density lipoprotein, Sf9 cells Apolipoprotein B-100’ün %17 N-Terminal bölgesinin klonlanması, anlatımı, saflaştırılması ve kantifikasyonu Özet Apolipoprotein B-100 (apo B) normal insan düşük yoğunluklu lipoprotein (LDL)’in yegane protein Artmış komponentidir. LDL düzeyleri ateroskleroz ve diğer koroner arter hastalıklarla ilişkilendirilmiştir. Apo B’nin büyüklüğü (4536 aa) yapısal olarak olmuş organize domeynlere karşılık gelen parçalar halinde çalışılmasını gerektirmektedir. ApoB’nin N-terminal bölgesinin %17’si olan B17, çok özel yapısal özellikleri olan bu domeynlerden biridir. Bu makale proteinin bu önemli parçasının klonlaması, anlatımı, saflaştırılması ve kantifikasyonu için protokolleri açıklamaktadır. Anahtar Sözcükler: Apolipoprotein (Apo B), C127 hücreleri, klonlama, düşük yoğunluklu lipoprotein (LDL), Sf9 hücreleri. Introduction Standing as one of the largest known proteins known, apolipoprotein B100 is the sole protein constituent of LDL (Mahley et al., 1984). The entire protein is a single peptide chain composed of 4536 amino acid residues, plus a 27 amino acid signal sequence. When glycosylated, the protein has a molecular mass of ~550 kDa, but the mature de-glycosylated chain is 512,937 Da. (Chen et al., 1986; Cladaras et al., 1986; Knott et al., 1986; Law et al., 1986; Yang et al., 1986). Apo B100 is synthesized in the liver and packaged into VLDL within the inner leaflet of the endoplasmic reticulum (Olofsson et al., 1987; Pease et al., 1991). Apo B100 has 25 cysteine residues of which sixteen form disulfide bonds (Yang et al., 1990; Shelness and Thornburg, 1996; Huang and Shelness, 1997). Except for the Cys-1/Cys-3 and Cys-2/Cys-4 bridges, all the other disulfide bonds occur between neighboring cysteines. All of the disulfide bonds occur in regions that are releasable
38 Hassan M. KHACHFE and David ATKINSON from the particle by trypsin digestion (Yang et al., 1989). Because of the size and insolubility of apo B, determination of its structural motifs responsible for the lipid association has been so difficult that only indirect probing has been done on this nonexchangeable protein. Biochemical and biophysical techniques, as well as computer algorithms have been used to study the domain structure and rearrangements of apo B. These studies have deepened our understanding of the domain arrangement of this huge protein. Therefore, it is necessary to study the structure of the protein in pieces, perhaps corresponding to structural or functional domains. For this reason, genetically engineered truncated forms have been obtained to study the domain organization in the protein. The N-terminal portion of the protein posed as an interesting candidate for structural studies for several reasons: 1) The striking homology it shows with other lipid transporting proteins, e.g., lipovitellin, whose structure was solved and studied vis-à-vis its function, and therefore, opened the door for computer modeling of the structure of apo B (Al- Ali and Khachfe, 2007). 2) This portion of the protein shows an optimal interaction with the microsomal triglyceride transfer protein (MTP). The presence of MTP complexed to the protein disulfide isomerase (PDI) found in the endoplasmic reticulum is an absolute requirement for the assembly of neutral lipids and phospholipids into chylomicrons and VLDL particles (Hussain et al., 1997). 3) Although truncated, this part of the protein is readily associated with a variety of phospholipids to from large discoidal particles (Herscovitz et al., 2001), and has interesting metabolic behaviors based on its glycosylation state, such that the Q158N mutation of the single glycosylation site in this domain decreases the secretion of the protein, but has little effect on its synthesis or its intracellular distribution (Vukmirica et al., 2002). 4) As mentioned above, the fact that seven out of the eight disulfide bonds found in apo B100 are located in the N-terminal domain suggest that this portion is compact, highly organized, and most likely globular (Prassl and Laggner, 2009). Hence, the 17% N-terminal domain of apo B100 was expressed with an aim to later characterize its structure and eventually relate to its function in the full-length protein. One of the restriction enzymes used to cut the apo B100 gene yielded a portion that corresponds to the 17% N-terminal part of the protein (Herscovitz et al., 1991). Following the same nomenclature process that described the different truncated forms of apo B100, this portion of the protein that corresponded to the N-terminal 17% of the full-length protein was then called apo B17, or simply B17. Materials and methods Materials Murine mammary carcinoma cells (C127) overexpressing B17 were obtained from Dr. V. Zannis (BUMC, Medicine) (Cladaras et al., 1987). Sf9 cells were from Life Technologies (Gaithersburg, MD). Baculovirus particles cloned with the B17 gene were a kind gift from Dr. G. Carraway. Dulbecco’s modified Eagle’s medium (DMEM), Sf-900 II serum-free medium (SFM), bovine fetal serum (BFS), penicillin / streptomycin (PS), and trypsin-EDTA were from Life Technologies (Gaithersburg, MD). N-Acetyl-Lleucinyl-L-leucinyl-L-norleucine (ALLN), aprotinin, leupeptin, phenyl-methyl-sulfonylfluoride (PMSF), sodium azide, and ethylenediamine tetraacetate (EDTA) were from Sigma (St. Louis, MO). Broad range protein marker (6,500 – 200,000 MW) was from BioRad (Hercules, CA). Gelatin Sepharose and Protein-G Sepharose 4 Fast Flow were from Pharmacia Amersham (Piscataway, NJ). Polyclonal goat anti-human apo B IgG, alkaline phosphatase-conjugated rabbit antigoat IgG, and horse-radish peroxidase (HRP)conjugated rabbit anti-goat IgG were from BioDesign (Saco, ME). Polyclonal sheep antihuman apo B IgG was from Roche Molecular Biochemicals (Indianapolis, IN). C-127 cells were permanently transfected with the gene coding for B17 as previously described (Claderas et al., 1987; Herscovitz et al., 1991). Mass expression of the protein was achieved using roller bottles (Claderas et al., 1987) or a Verax System-1 Bioreactor (Verax, Lebanon, NH) that was modified – in-house – and coupled with a ceramic core reactor, which eventually increased the number of cells to near tissue density while automating the media feed and harvest processes. The harvested or stored media were then vacuum-filtered through 0.45 µm pore size filter paper, and then concentrated 25-fold using an Amicon stirred-cell with a 30,000 MWCO membrane. The concentrate was processed for protein purification. Sf9 – Spodoptera frugiperda – insect cells adapted to serum-free suspension culture in Sf-900
Page 1 and 2: Journal of Cell and Molecular Biolo
Page 3 and 4: Haliç University Faculty of Arts a
Page 8 and 9: Öyle ki, biyolojik ritimler tek h
Page 10 and 11: profilindeki 24 saatlik değişikli
Page 12 and 13: Sirkadiyan ritim genleri ve kanser
Page 14 and 15: and cancer. Cancer Res. 65 (15): 68
Page 18 and 19: argued that Ca 2+ transfer was more
Page 20 and 21: chemotherapy which has many differe
Page 22 and 23: Kadiu I and Gendelman H. Human Immu
Page 26 and 27: determined. The frequency of rare a
Page 28 and 29: Turkish barley cultivar screening w
Page 30 and 31: Moreover, correspondence analysis b
Page 34 and 35: No studies on the genotoxic effect
Page 36 and 37: group of acute treatment, this valu
Page 38 and 39: indicated in in vitro studies. Furt
Page 40: Marie PJ. Strontium ranelate: A nov
Page 45 and 46: 40 Hassan M. KHACHFE and David ATKI
Page 47 and 48: 42 Hassan M. KHACHFE and David ATKI
Page 49 and 50: 44 Emmanuel AMLABU et al. Introduct
Page 51 and 52: 46 Emmanuel AMLABU et al. Purificat
Page 53 and 54: 48 Emmanuel AMLABU et al. 1/V (µmo
Page 58 and 59: Maintenance of Alternaria alternata
Page 60 and 61: exhibited minimum cellulase activit
Page 64 and 65: Submission Policies and Authorship
Page 67: Journal of Cell and Volume 9 · No

Journal of Cell and Molecular Biology - ResearchGate

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?