University of Illinois Urbana-Champaign

Expression, Purification, and Crystallization Screening of a PCAT Transporter from Ornithinibacillus bavariensis

Im, Jamie

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https://hdl.handle.net/2142/133270

Description

Title
Expression, Purification, and Crystallization Screening of a PCAT Transporter from Ornithinibacillus bavariensis
Author(s)
Im, Jamie
Issue Date
2026-05-14
Director of Research (if dissertation) or Advisor (if thesis)
Nair, Satish
Department of Study
School of Chemical Sciences
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana Champaign
Degree Name
B.S. (bachelor's)
Degree Level
Thesis
Date of Ingest
2026-05-14T01:13:19-05:00
Keyword(s)
PCAT, OrgT, ABC Transporter, RiPP
Language
eng
Abstract
Peptidase-containing ATP-binding cassette transporters (PCATs) are bifunctional enzymes that process and export peptide substrates in Gram-positive bacteria. Canonical PCAT substrates are typically cleaved at a double-glycine (GG) motif. However, the mechanism for how PCATs recognize and tolerate non-canonical motifs is unclear. OrgT from Ornithinibacillus bavariensis is observed to recognize a non-canonical GK motif. Since lysine is larger than glycine and positively charged, this substitution is expected to be incompatible with the currently established active-site geometry. To investigate this phenomenon, we aimed to purify and crystallize OrgT to evaluate its cocrystal structure with its substrate. We initially found that BL21 cells grown in LB produced the best OrgT expression through an expression check. Afterward, repeated purification trials showed that OrgT yield was best at pH 8.6 and 300 mM NaCl. Although aggregation was minimized under the aforementioned conditions, crystallization screening trials showed limited precipitation, indicating the need for a higher concentration. These results provide the groundwork and omit possible conditions for future crystallographic studies of OrgT.
Type of Resource
text

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