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1. Zhang, Na. Folding Analysis of Reduced Bovine Pancreatic Trypsin Inhibitor (BPTI) with Aromatic Thiols and Disulfides In Vitro.

Degree: PhD, Biochemistry, 2018, Florida International University

Almost all therapeutic proteins contain disulfide bonds to stabilize their native structure. Recombinant DNA technology enables many therapeutic proteins to be produced in bacteria, but the expression of native proteins is not always efficient due to the limited ability of bacteria to form disulfide bonds in vivo. It is often necessary to employ in vitro oxidative folding process to form the native disulfide bonds to obtain the native structure of disulfide-containing proteins. Aromatic disulfides are small molecules designed to match some of the physical properties of the active site of protein disulfide isomerase (PDI), which catalyzes the folding process of disulfide-containing proteins in eukaryotes. Three aromatic thiols with varying charges, PA, SA and QAS thiol, were used to fold reduced BPTI in vitro. Bovine pancreatic trypsin inhibitor (BPTI) is positively charged (pI = 10.5) at pH 7.3, and we hypothesized that mixed disulfide intermediates formed between BPTI and negatively charged small molecule thiols were more likely to precipitate due to their minimized net charge. Protein precipitation was observed during folding with negatively charged thiols, PA and SA, but not positively charged thiol QAS. At the folding pH of 7.3, almost 90% of native BPTI was produced in 2 h with the conditions of 0.25 mM QAS disulfide and 10 mM QAS thiol. Only 25% of native BPTI was produced in 2 h with the best conditions for glutathione and glutathione disulfide. Aromatic thiols with an elongated alkyl group on the aromatic ring, butyl, hexyl and octyl thiol, were hypothesized to increased interactions with the hydrophobic core of disulfide-containing proteins during folding, allowing more facile access to buried disulfide bonds. However, the longer the hydrocarbon chain, the more likely protein precipitation was to occur. About 90% native BPTI was formed in 1 h with 0.25 mM hexyl disulfide and 10 mM hexyl thiol. A method using capillary electrophoresis (CE) to analysis the oxidative folding process of reduced BPTI with small molecule thiols and disulfides was also developed. Folding of reduced BPTI with QAS disulfide was analyzed using CE in a shorter run time. The consumption of protein samples and solvent solutions was minimized. Advisors/Committee Members: Watson J. Lees, Bruce R. McCord, Xiaotang Wang, Bernard S. Gerstman.

Subjects/Keywords: Protein folding; Aromatic thiols; BPTI; HPLC; CE; Biochemistry, Biophysics, and Structural Biology; Biotechnology

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Zhang, N. (2018). Folding Analysis of Reduced Bovine Pancreatic Trypsin Inhibitor (BPTI) with Aromatic Thiols and Disulfides In Vitro. (Doctoral Dissertation). Florida International University. Retrieved from https://digitalcommons.fiu.edu/etd/3903 ; 10.25148/etd.FIDC007004 ; FIDC007004

Chicago Manual of Style (16th Edition):

Zhang, Na. “Folding Analysis of Reduced Bovine Pancreatic Trypsin Inhibitor (BPTI) with Aromatic Thiols and Disulfides In Vitro.” 2018. Doctoral Dissertation, Florida International University. Accessed September 22, 2020. https://digitalcommons.fiu.edu/etd/3903 ; 10.25148/etd.FIDC007004 ; FIDC007004.

MLA Handbook (7th Edition):

Zhang, Na. “Folding Analysis of Reduced Bovine Pancreatic Trypsin Inhibitor (BPTI) with Aromatic Thiols and Disulfides In Vitro.” 2018. Web. 22 Sep 2020.

Vancouver:

Zhang N. Folding Analysis of Reduced Bovine Pancreatic Trypsin Inhibitor (BPTI) with Aromatic Thiols and Disulfides In Vitro. [Internet] [Doctoral dissertation]. Florida International University; 2018. [cited 2020 Sep 22]. Available from: https://digitalcommons.fiu.edu/etd/3903 ; 10.25148/etd.FIDC007004 ; FIDC007004.

Council of Science Editors:

Zhang N. Folding Analysis of Reduced Bovine Pancreatic Trypsin Inhibitor (BPTI) with Aromatic Thiols and Disulfides In Vitro. [Doctoral Dissertation]. Florida International University; 2018. Available from: https://digitalcommons.fiu.edu/etd/3903 ; 10.25148/etd.FIDC007004 ; FIDC007004

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