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Several truncated forms of apo B100 have been identified in the plasma of human subjects, the shortest of which was denoted apo B31, corresponding to 31% portion of the full length protein (Havel, 1989; Young et al., 1990). Although shorter forms are indeed synthesized, they either don't find their way to the plasma because they are not secreted or, once in the plasma, they are rapidly degraded (Collins et al., 1988). The present study reaffirms previously reported results showing that shorter forms of apo B100, namely B17, can be secreted by C127 cells (Herscovitz et al., 1991) and Sf-9 cells (Choi et al., 1995). However, this study shows that the mass expression of B17 results in degradation products that are clearly correlated with the larger number of cells used in the expression system. The use of a protease inhibitor, PMSF, in the C127 cell system prevented the degradation, indicating that proteases in the media and/or inside the cells were responsible for this effect. On the other hand, addition of EDTA to the insect cell media to a final concentration of 20µM EDTA also prevented the degradation in that system. SDS-PAGE analysis of the expressed and purified proteins from both expression systems showed that the two products are similar in size. Further assessment using CD spectroscopy confirmed that the products expressed and purified from the two systems are identical in terms of their structural contents. Discussion Apolipoprotein B100 (apo B) is the only protein found on human low density lipoprotein (LDL) particles. LDL is the agent provocateur for atherosclerosis and other coronary heart diseases. Apo B is a large (4536 amino acids, 550 kDa) secretory glycoprotein that has unique structural properties. The large size of apo B necessitated that it be studied in pieces corresponding to its structurally organized domains. In the present work, we studied the conformational and stability properties of the 17% N-terminal domain of apo B, B17. This portion of the protein is secreted predominantly lipid-free, and plays an important role in the initiation and assembly of the LDL particle (Herscovitz et al., 2001). Mass expression of B17 was achieved via two different cell lines: Mammalian and insect. The mammalian-derived murine C127 cells were transfected with a bovine papilloma virus-based expression vector, while the insect-derived Sf-9 Expression, purification and quantification of B17 41 cells were transfected with a baculovirus-based expression vector. Previously reported methods and protocols were enhanced and fine-tuned to overcome a degradation problem associated with the mass expression of the protein. The protein yields from both systems were compared for purity and homogeneity, and were found to be identical. Acknowledgement The initial C127 cell batch and the initial baculovirus particles were kind gifts from Drs. V. Zannis and G. Carraway, respectively. This project was partially supported by an award from the National Health Institutes (NIH). References Chen SH, Yang CY, Chen PF, Setzer D, Tanimura M, Li WH, Gotto AM and Chan L. The Complete cDNA and Amino Acid Sequence of Human Apolipoprotein B-100. J Biol Chem. 261(28): 12918-12921, 1986. Choi SY, Sivaram P, Walker DE, Curtiss LK, Gretch DG, Sturley SL, Attie AD, Deckelbaum RJ and Goldberg IJ. Lipoprotein lipase association with lipoproteins involves proteinprotein interaction with apolipoprotein B. J Biol Chem. 270(14):8081-8086, 1995. Cladaras C, Hadzopoulou-Cladaras M, Felber BK, Pavlakis G and Zannis VI. The molecular basis of a familial apoE deficiency. An acceptor splice site mutation in the third intron of the deficient apoE gene. J Biol Chem. 262(5):2310- 2315, 1987. Cladaras C, Hadzopoulou-Cladaras M, Nolte RT, Atkinson D and Zannis VI. The Complete Sequence and Structural Analysis of Human Apolipoprotein B-100: Relationship Between apoB-100 and apoB-48 Forms. EMBO J. 5: 3495-3506, 1986. Collins DR, Knott TJ, Pease RJ, Powell LM, Wallis Simon C, Robertson S, Pullinger CR, Milne RW, Marcel YL, Humphries SE, Talmud PJ, Lloyd JK, Miller NE, Muller D and Scott J. Truncated Variants of Apolipoprotein B Cause Hypobetalipoproteinemia. Nucleic Acids Research. 16(17): 8361-8375, 1988. Havel RJ. Biology of cholesterol, lipoproteins and atherosclerosis. Clin Exp Hypertens A. 11(5- 6):887-900, 1989. Herscovitz H, Derksen A, Walsh MT, McKnight CJ, Gantz DL, Hadzopoulou-Cladaras M,
42 Hassan M. KHACHFE and David ATKINSON Zannis V, Curry C, Small DM. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins. J Lipid Res. 42(1):51-59, 2001. Herscovitz H, Hadzopoulou-Cladaras M, Walsh, MT, Cladaras C; Zannis VI and Small DM. Expression, secretion, and lipid-binding characterization of the N- terminal 17% of apolipoprotein B. Proc Natl Acad Sci U S A. 88(20): 9375, 1991. Huang XF and Shelness GS. Identification of cysteine pairs within the amino-terminal 5% of apolipoprotein B essential for hepatic lipoprotein assembly and secretion. J Biol Chem. 272(50):31872-31876, 1997. Hussain MM, Bakillah A and Jamil H. Apolipoprotein B binding to microsomal triglyceride transfer protein decreases with increases in length and lipidation: implications in lipoprotein biosynthesis. Biochemistry. 36 (42):13060-13067, 1997. Al-Ali H, Khachfe H. The N-Terminal Domain of Apolipoprotein B-100: Structural Characterization by Homology Modeling. BMC Biochemistry. 8:12, 2007. Knott TJ, Pease RJ, Powell LM, Wallis SC, Rall SC Jr, Innerarity TL, Blackhart B, Taylor WH, Marcel Y, Milne R, Johnson D, Fuller M, Luisi AJ, McCarthy BJ, Mahley RW, Levy-Wilson B and Scott J. Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature. 323(6090): 734-738, 1986. Law SW, Grant SM, Higuchi K, Hospattankar A, Lackner K, Lee N and Brewer HB Jr. Human Liver Apolipoprotein B-100 cDNA: Complete Nucleic Acid and Derived Amino Acid Sequence. Proc Nat. Acad Sci. USA. 83: 8142- 8146, 1986. Lowry OH, Rosenbrough NJ, Farr AL and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 193:265-75, 1951. Mahley RW and Angelin B. Type III Hyperlipoproteinemia: Recent Insights into the Genetic Defect of Familial Dysbetalipoproteinemia. Advance in Internal Medicine: Year Book Med Publ Inc. 29: 385- 411, 1984. Olofsson SO, Bostrom K, Carlsson P, Boren J, Wettesten M, Bjursell G, Wiklund O and Bondjers G. Structure and biosynthesis of apolipoprotein B Am Heart J. 113(2 Pt 2):446- 52, 1987. Pease RJ, Harrison GB and Scott J. Cotranslocational insertion of apolipoprotein B into the inner leaflet of the endoplasmic reticulum. Nature. 353(6343):448-450, 1991. Prassl R and Laggner P. Molecular structure of low density lipoprotein: current status and future challenges. Eur Biophys J. 38:145–158, 2009. Shelness GS and Thornburg JT. Role of intramolecular disulfide bond formation in the assembly and secretion of apolipoprotein B- 100-containing lipoproteins. J Lipid Res. 37:408-419, 1996. Vuk-mirica, J, Nishimaki-Mogami T, Tran K, Shan J, McLeod RS, Yuan J and Yao Z. The N-linked oligosaccharides at the amino terminus of human apoB are important for the assembly and secretion of VLDL. J Lipid Res. 43:1496–1507, 2002. Yang CY, Gu ZW, Kim TW, Chen, SH, Pownall HJ, Sharp PM, Liu SW, Li WH, Gotto AM Jr and Chan L. Structure of Apolipoprotein B-100 of Human Low Density Lipoproteins. Arteriosclerosis. 9, 96-108, 1989. Yang CY, Kim TW, Weng SA, Lee B, Yang M and Gotto AM. Isolation and Characterization of Sulfhydryl and Disulfide Peptides of Human Apolipoprotein B-100. Proc. Natl. Acad. Sci. USA. 87: 5523-5527, 1990. Yang CY, Yang T, Pownall HJ and Gotto AM Jr. The primary structure of apolipoprotein A-I from rabbit high-density lipoprotein. Eur J Biochem. 160: 427-431, 1986. Young SG, Hubl, ST, Smith RS, Snyder, SM and Terdiman, JF. Familial hypobetalipoproteinemia caused by a mutation in the apolipoprotein B gene that results in a truncated species of apolipoprotein B (B-31): a unique mutation that helps to define the portion of the apolipoprotein B molecule required for the formation of buoyant, triglyceride-rich lipoproteins. J Clin Invest. 75:933-942, 1990.
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