Biology subjects

Lee, T. H.

Publications and source records attributed to Lee, T. H..

7 recordsLinked to original sources

bioRxiv2025

Combinatorial Protein Language Model-Guided Engineering of TEV Protease for Enhanced Stability and Production

Industrial enzyme engineering focuses on improvement of enzyme production yield, stability, catalytic activity, and substrate specificity, but often suffers from low efficiency with time-consuming and labor-intensive design and screening processes of massive libraries. Recent advances in AI and machine learning created protein language models trained by numerous datasets and shed new lights to speed up the enzyme engineering processes with high accuracy structural prediction. Here, we developed a highly efficient enzyme engineering strategy combining three protein language models (xTrimoMPNN-Thermo, ESM-IF, and MPNNsol) and use it to generate TEV protease variants with improved expression, stability, and function. The results indicated that a small number of TEV protease designs (<50 designs) were sufficient to develop variants with desired properties, demonstrating its high efficiency. Our strategy could be broadly applied to accelerate designing and engineering various industrial enzymes.

bioengineering↗

bioRxiv2025

Computational Design of Soluble CCR8 Analogues with Preserved Antibody Binding

G protein-coupled receptors (GPCRs) represent the largest class of drug targets, yet their membrane-embedded nature poses significant challenges for structural studies and therapeutic development. Here, we report the successful computational design and experimental validation of soluble CCR8 analogues that maintain native antibody binding properties. Using an integrated pipeline combining ProteinMPNN-sol sequence design, and structure-based filtering, we generated 13 CCR8 analogues from 272 initial designs across three N-terminal truncation strategies. Experimental validation confirmed 62% success rate (8/13 designs) with protein yields of 1.19-73.72 mg/L in aqueous buffer, representing a significant improvement over traditional membrane protein production method. Surface plasmon resonance analysis demonstrated that all analogues retained mAb1 binding with dissociation constants ranging from 77-857 nM, comparable to wild-type CCR8 (KD = 190 nM). Despite extensive sequence divergence (10-13% identity with wild-type CCR8), structural integrity was preserved as evidenced by binding affinity maintenance and computational structural validation. This work demonstrates the feasibility of computationally designing functional soluble analogues of challenging membrane proteins, with implications for accelerating drug discovery, antibody development, and structural biology studies. Our approach addresses critical limitations in membrane protein accessibility while preserving native epitope presentation, opening new avenues for therapeutic target characterization and binder discovery.

bioengineering↗

bioRxiv2022

Discovery and biosynthesis of imidazolium antibiotics from a probiotic Bacillus licheniformis

Antibiotic resistance is one of the worlds most urgent public health problems and therefore novel antibiotics to kill drug-resistant bacteria are desperately needed. So far, natural product-derived small molecules have been the major sources for new antibiotics. Here we describe a family of antibacterial metabolites isolated from a probiotic bacterium Bacillus licheniformis. Cross-streaking assay followed by activity-guided isolation yielded a novel antibacterial metabolite bacillimidazole G, which possesses a rare imidazolium ring in the structure, showing MIC values of 0.7-2.6 g/mL against human pathogenic Gram-positive and Gram-negative bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and a lipopolysaccharide(LPS)-lacking Acinetobacter baumannii {Delta}lpxC. Bacillimidazole G also lowered MICs of colistin, a Gram-negative antibiotic, up to 8-fold against wild-type E. coli MG1655 and Acinetobacter baumannii. We propose biosynthetic pathway of the characterized metabolites based on the precursor-feeding studies, chemical biological approach, biomimetic total synthesis, and biosynthetic genes knockout method. TOC/Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=34 SRC="FIGDIR/small/511033v1_ufig1.gif" ALT="Figure 1"> View larger version (8K): org.highwire.dtl.DTLVardef@184f545org.highwire.dtl.DTLVardef@2a69eorg.highwire.dtl.DTLVardef@8cf13org.highwire.dtl.DTLVardef@530001_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

New Genotypes and Diversity of Orientia tsutsugamushi Isolated from Scrub Typhus Patients in South Korea as Determined by Multilocus Sequence Typing

Orientia tsutsugamushi, an obligate intracellular organism, is the causative agent of scrub typhus, which is endemic in the Asia-Pacific region. No comparative studies on the genotypic properties of O. tsutsugamushi have been performed using multilocus sequence typing (MLST) in South Korea. Here, we characterized 51 clinical isolates from Jeonju, in southwestern Korea, and we compared them to isolates from Thailand, Laos, and Japan. We also identified 10 new alleles and six novel sequence types. Overall, our results suggest that the relative genetic stability and the clonal populations of O. tsutsugamushi strains in South Korea have remained mostly conserved. Author summaryScrub typhus is a life-threatening disease, caused by infection with O. tsutsugamushi, a Gram-negative intracellular bacterium. Approximately one million people are infected globally every year, especially in the Asia-Pacific region. Strains of O. tsutsugamushi are typically distinguished serologically on the basis of sequences of the highly polymorphic 56-kDa outer membrane protein. Multilocus sequence typing (MLST) is a generic typing method that provides a unified bacterial isolate characterization approach that can be used for evolutionary and population studies of bacteria. In this study, we describe the development and application of a MLST scheme that was applied to 51 O. tsutsugamushi isolates. We found 10 new alleles and six new STs, which yielded a total of seven O. tsutsugamushi STs in South Korea. Among seven different STs (ST 48, 93-98), ST 48 account for the largest proportion (49.0%) of O. tsutsugamushi STs in South Korea. With the exception of the appearance of six novel STs, the clonal populations have remained conserved but further study of population structure and evolutionary trends is warranted.

microbiology↗

bioRxiv2020

PCNA monoubiquitination is regulated by diffusion of Rad6/Rad18 complexes along RPA filaments

Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the PCNA sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA damaging agents and is tightly regulated to restrict TLS to damaged DNA. RPA, the major single strand DNA (ssDNA) binding protein, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18*RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination.

biochemistry↗

bioRxiv2017

Dominance of ST131 Escherichia coli carrying blaCTX-M in patients with bloodstream infections caused by cephalosporin-resistant strains in Australia, New Zealand and Singapore: whole genome analysis of isolates from a randomised trial

Synopsis/AbstractO_ST_ABSObjectivesC_ST_ABSTo characterise multi-drug resistant Escherichia coli isolated from patients in Australia, New Zealand and Singapore with bloodstream infection (BSI).\n\nMethodsWe prospectively collected third-generation cephalosporin resistant (3GC-R) E. coli from blood cultures obtained from patients enrolled in a randomised controlled trial. Whole genome sequencing was used to characterise antibiotic resistance genes, sequence types (STs), plasmids and phylogenetic relationships. Antibiotic susceptibility was determined using disk diffusion and Etest.\n\nResultsA total of 70 E. coli were included, of which the majority were ST131 (61.4%). BSI was most frequently from a urinary source (69.6%), community-associated (62.9%) and in older patients (median age 71 years [IQR 64-81]). The median Pitt bacteraemia score at presentation was 1 (IQR 0-2, range 0-3) and ICU admission was infrequent (3.1%). ST131 possessed significantly more acquired resistance genes than non-ST131 (p=0.003). Clade C1/C2 ST131 predominated (30.2% and 53.5% of all ST131 respectively) and these were all resistant to ciprofloxacin. All clade A ST131 were community-associated. The predominant ESBL types were blaCTX-M (78.6% of isolates) and were strongly associated with ST131, with the majority blaCTX-M-15. Clade C1 was associated with blaCTX-M-14 and blaCTX-M-27, whereas blaCTX-M-15 predominated in clade C2. Plasmid-mediated AmpC (p-AmpC) genes (mainly blaCMY-2) were also frequent (17.1%) but were more common with non-ST131 strains (p< 0.001). The majority of plasmid replicon types were IncF.\n\nConclusionsIn a prospective collection of 3GC-R E. coli causing BSI in the Australasian region, community-associated Clade C1/C2 ST131 predominate in association with blaCTX-M ESBLs, although a significant proportion of non-ST131 strains carried blaCMY-2.

microbiology↗

bioRxiv2016

PRDX6 inhibits neurogenesis of neural precursor cells through downregulation of wdfy1 mediated TLR4 signal

Impaired neurogenesis has been associated with brain disorders. The role of peroxiredoxin 6 (PRDX6) in the neurodegenerative diseases is very controversial. To demonstrate the role of PRDX6 in neurogenesis, we compared neurogenesis ability and studied the molecular mechanisms. It was found that the neurogenesis of neural stem cells and expression of the marker protein were lowered in PRDX6 Tg-mice compared with non-tg mice. Moreover, the expression of wdfy1 was dramatically decreased in PRDX6-Tg mice, also, we observed that wdfy1 siRNA decreases the differentiation ability of primary neural stem cells to astrocyte and neuronal cells as well as PC12 cells. However, knockdown of PRDX6 recovered neurogenesis in the brain of PRDX6-Tg mice as well as PC-12 cells. We also showed that TLR4 was dramatically reduced in PRDX6 Tg mice as well as PC-12 cells and PRDX6 overexpression reduced neurogenesis was rescued after treatment of TLR4 siRNA. We further found that reduced TLR4 expression and neurogenesis was reversed in the neuron from PRDX6-Tg mice as well as PC12 cells by introduction of wdfy1 plasmid. Moreover, TLR4 siRNA reduced neurogenesis and wdfy1 expression. This study indicated that PRDX6 inhibits neurogenesis of neural precursor cells through TLR4 dependent downregulation of wdfy1.

developmental biology↗