Trehalose Glycopolymers as Excipients for Protein Stabilization

被引:122
作者
Lee, Juneyoung [1 ,2 ]
Lin, En-Wei [1 ,2 ]
Lau, Uland Y. [3 ]
Hedrick, James L. [1 ,2 ]
Bat, Erhan [1 ,2 ]
Maynard, Heather D. [1 ,2 ,3 ]
机构
[1] Univ Calif Los Angeles, Dept Chem & Biochem, Los Angeles, CA 90095 USA
[2] Univ Calif Los Angeles, Calif Nanosyst Inst, Los Angeles, CA 90095 USA
[3] Univ Calif Los Angeles, Dept Bioengn, Los Angeles, CA 90095 USA
基金
美国国家科学基金会;
关键词
Monomers;
D O I
10.1021/bm4003046
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Herein, the synthesis of four different trehalose glycopolymers and investigation of their ability to stabilize proteins to heat and lyophilization stress are described. The disaccharide, alpha,alpha-trehalose, was modified with a styrenyl acetal, methacrylate acetal, styrenyl ether, or methacrylate moiety resulting in four different monomers. These monomers were then separately polymerized using free radical polymerization with azobisisobutyronitrile (AIBN) as an initiator to synthesize the glycopolymers. Horseradish peroxidase and glucose oxidase were incubated at 70 and 50 degrees C, respectively, and beta-galactosidase was lyophilized multiple times in the presence of various ratios of the polymers or trehalose. The protein activities were subsequently tested and found to be significantly higher when the polymers were present during the stress compared to no additive and to equivalent amounts of trehalose. Different molecular weights (10 kDa, 20 kDa, and 40 kDa) were tested, and all were equivalent in their stabilization ability. However, some subtle differences were observed regarding stabilization ability between the different polymer samples, depending on the stress. Small molecules such as benzyl ether trehalose were not better stabilizers than trehalose, and the trehalose monomer decreased protein activity, suggesting that hydrophobized trehalose was not sufficient and that the polymeric structure was required. In addition, cytotoxicity studies with NIH 3T3 mouse embryonic fibroblast cells, RAW 264.7 murine macrophages, human dermal fibroblasts (HDFs), and human umbilical vein endothelial cells (HUVECs) were conducted with polymer concentrations up to 8 mg/mL. The data showed that all four polymers were noncytotoxic for all tested concentrations. The results together suggest that trehalose glycopolymers are promising as additives to protect proteins from a variety of stressors.
引用
收藏
页码:2561 / 2569
页数:9
相关论文
共 48 条
[1]   Influence of preparation procedure on polymer composition:: synthesis and characterisation of polymethacrylates bearing β-D-glucopyranoside and β-D-galactopyranoside residues [J].
Ambrosi, M ;
Batsanov, AS ;
Cameron, NR ;
Davis, BG ;
Howard, JAK ;
Hunter, R .
JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 1, 2002, Royal Society of Chemistry (01) :45-52
[2]   Biotechnology applications of amino acids in protein purification and formulations [J].
Arakawa, T. ;
Tsumoto, K. ;
Kita, Y. ;
Chang, B. ;
Ejima, D. .
AMINO ACIDS, 2007, 33 (04) :587-605
[3]   THE STABILIZATION OF PROTEINS BY OSMOLYTES [J].
ARAKAWA, T ;
TIMASHEFF, SN .
BIOPHYSICAL JOURNAL, 1985, 47 (03) :411-414
[4]   Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase [J].
Asad, Sedigheh ;
Torabi, Seyed-Fakhreddin ;
Fathi-Roudsari, Mehrnoosh ;
Ghaemi, Nasser ;
Khajeh, Khosro .
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 2011, 48 (04) :566-570
[5]   Structural thermodynamics of protein preferential solvation: Osmolyte solvation of proteins, aminoacids, and peptides [J].
Auton, Matthew ;
Bolen, D. Wayne ;
Rosgen, Jorg .
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 2008, 73 (04) :802-813
[6]   Nonionic Homopolymeric Amphipols: Application to Membrane Protein Folding, Cell-Free Synthesis, and Solution Nuclear Magnetic Resonance [J].
Bazzacco, Paola ;
Billon-Denis, Emmanuelle ;
Sharma, K. Shivaji ;
Catoire, Laurent J. ;
Mary, Sophie ;
Le Bon, Christel ;
Point, Elodie ;
Baneres, Jean-Louis ;
Durand, Gregory ;
Zito, Francesca ;
Pucci, Bernard ;
Popot, Jean-Luc .
BIOCHEMISTRY, 2012, 51 (07) :1416-1430
[7]   Trapping and Stabilization of Integral Membrane Proteins by Hydrophobically Grafted Glucose-Based Telomers [J].
Bazzacco, Paola ;
Sharma, K. Shivaji ;
Durand, Gregory ;
Giusti, Fabrice ;
Ebel, Christine ;
Popot, Jean-Luc ;
Pucci, Bernard .
BIOMACROMOLECULES, 2009, 10 (12) :3317-3326
[8]   Protein stability during freezing: Separation of stresses and mechanisms of protein stabilization [J].
Bhatnagar, Bakul S. ;
Bogner, Robin H. ;
Pikal, Michael J. .
PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY, 2007, 12 (05) :505-523
[9]   Effects of naturally occurring osmolytes on protein stability and solubility: issues important in protein crystallization [J].
Bolen, DW .
METHODS, 2004, 34 (03) :312-322
[10]   Relationship between conformational stability and lyophilization-induced structural changes in chymotrypsin [J].
Carrasquillo, KG ;
Sanchez, C ;
Griebenow, K .
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY, 2000, 31 (31) :41-53