Poster B5 - Heterologous expression of engineered titin immunoglobulin domain mutants for sequence-property relationship analysis

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Huamin Gao

Undergraduate Researcher
University of Illinois Urbana Champaign
St. Louis, Missouri, United States

Co-Author(s)

SS

Shri Subramani

PhD Candidate
Washington University in St. Louis, Department of Energy, Environmental and Chemical Engineering, United States

Primary Investigator(s)

FZ

Fuzhong Zhang

Professor
Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, United States

Introduction: There is a need to produce synthetic, sustainable biomaterials for various commercial and research applications as many natural counterparts are difficult to obtain and mass produce. Titin is a promising material which is an ultra-high molecular weight (UHMW) protein found in animal muscle fiber comprising of highly repetitive amino acid sequences. Titin has potential in a wide range of biomedical applications, including scaffolding for tissue engineering, skeletal muscle regeneration, and drug delivery. Previous studies have recombinantly synthesized UHMW titin from the immunoglobin (Ig) domains 67-70 of rabbit soleus titin, but the relationship between amino acid sequences and mechanical properties remains poorly understood (1). To better understand this relationship, various titin mutants have been identified that are predicted to have different mechanical properties than the original rabbit titin Ig67-70. However, work needs to be done to confirm the expression of such mutants, which is the focus of this project.

Materials and

Methods: E. Coli strain NEB10β was transformed with plasmids encoding different titin mutant sequences. Transformed cells were cultured first in Luria Broth (LB) media (10g/L tryptone, 5g/L yeast extract, 10g/L glucose, and 10g/L NaCl) with the appropriate antibiotic overnight at 37°C on an orbital shaker, then transferred to Terrific Broth (TB) media (24g/L yeast extract, 20g/L tryptone, 10mL/L glycerol, 2.3g/L KH2PO4, and 12.5g/L K2HPO4). The cultures were induced with 0.4% Arabinose once the optical density (OD) at 600 nm reached 3. Induced cultures grew overnight, and cells were harvested by centrifugation. The pellets were resuspended in 6M guanidine-HCl buffer to lyse the cells. The cell lysate was filtered and purified using a Ni-NTA column. 8M Urea buffer with 0-50mM Imidazole was used to wash the column before the desired protein was eluted with 300mM imidazole. Purified proteins were dialyzed in 1% acetic acid, freeze-dried, weighed, then stored at -80°C for future mechanical testing. This process is shown in Figure 1.

Results, Conclusions, and Discussions: Four titin mutants were successfully expressed in E. Coli with an average titer of 50 mg/L. The obtained proteins will be fabricated into fibers for later mechanical testing. The successful expression of titin mutants achieved in this project opens more sustainable production for such fibers through synthetic pathways and allows for later testing to be done to identify a relationship between titin’s amino acid sequence and its mechanical properties. After such a relationship is established, titin’s mechanical properties may be tuned to be utilized in a wide variety of medical and industrial applications.

Acknowledgements (Optional): Acknowledgements:
We would like to thank CEMB and SMARC at the Washington University in St. Louis for their support.

References:
(1) Bowen, C.H., Sargent, C.J., Wang, A. et al. Microbial production of megadalton titin yields fibers with advantageous mechanical properties. Nat Commun 12, 5182 (2021). https://doi.org/10.1038/s41467-021-25360-6