دورية أكاديمية
LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding.
| العنوان: | LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding. |
|---|---|
| المؤلفون: | Mengwasser KE; Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom., Bryant CE, Gay NJ, Gangloff M |
| المصدر: | Protein expression and purification [Protein Expr Purif] 2011 Apr; Vol. 76 (2), pp. 173-83. Date of Electronic Publication: 2010 Dec 02. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Academic Press Country of Publication: United States NLM ID: 9101496 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-0279 (Electronic) Linking ISSN: 10465928 NLM ISO Abbreviation: Protein Expr Purif Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: Orlando, FL : Academic Press Original Publication: San Diego : Academic Press, c1990- |
| مواضيع طبية MeSH: | Culture Media*, Antigens, Surface/*biosynthesis , Lipopolysaccharides/*pharmacology , Lymphocyte Antigen 96/*biosynthesis , Lymphocyte Antigen 96/*isolation & purification , Pichia/*metabolism , Recombinant Fusion Proteins/*biosynthesis, Animals ; Antigens, Surface/chemistry ; Antigens, Surface/genetics ; Antigens, Surface/isolation & purification ; Chromatography, Gel ; Cloning, Molecular ; Disulfides/chemistry ; Disulfides/metabolism ; Electrophoresis, Polyacrylamide Gel ; Horses ; Humans ; Ligands ; Lymphocyte Antigen 96/chemistry ; Lymphocyte Antigen 96/genetics ; Phenotype ; Pichia/chemistry ; Pichia/genetics ; Protein Multimerization ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/isolation & purification |
| مستخلص: | Myeloid differentiation proteins MD-1 and MD-2 have both been shown to form a heterogeneous collection of oligomers when expressed in absence of their respective receptor, RP105 and TLR4. The biological relevance of these oligomers is not clear. Only monomeric proteins have been found to be active and able to trigger an immune response to endotoxin by modulating the TLR4 pathway. In this study, we produced variants of MD-1 and MD-2 in Pichia pastoris. To minimize the time and expense of initial expression tests, small-scale cultures have been set up to allow the rapid identification of the highest expressing clone and the optimal expression conditions. The expression vectors used, the site of linearization and the locus of integration affected the yield of transformation. Next we screened culture additives and found that they significantly increased the fraction of monomeric proteins secreted in the culture medium (up to 15% of the total MD protein produced). We confirmed their presence by size-exclusion chromatography. Optimal anti-aggregation agents were protein-dependent except for LPS that presented stabilizing effects for all MD proteins. Contrary to previous reports, this study suggests that MD-1 can bind to LPS. (Copyright © 2010 Elsevier Inc. All rights reserved.) |
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| معلومات مُعتمدة: | T32 GM007753 United States GM NIGMS NIH HHS; RG47206 United Kingdom WT_ Wellcome Trust |
| المشرفين على المادة: | 0 (Antigens, Surface) 0 (Culture Media) 0 (Disulfides) 0 (LY86 protein, human) 0 (LY96 protein, human) 0 (Ligands) 0 (Lipopolysaccharides) 0 (Lymphocyte Antigen 96) 0 (Recombinant Fusion Proteins) |
| تواريخ الأحداث: | Date Created: 20101207 Date Completed: 20110531 Latest Revision: 20211020 |
| رمز التحديث: | 20231215 |
| مُعرف محوري في PubMed: | PMC3032050 |
| DOI: | 10.1016/j.pep.2010.11.018 |
| PMID: | 21130168 |
| قاعدة البيانات: | MEDLINE |
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