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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster U16 - Development of Strategies for Targeted Delivery and Retention Enhancement of Mitsugumin 53 for Tissue Regeneration

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Abria Granger (she/her/hers)

Student
University of Texas San Antonio
San Antonio, Texas, United States

Co-Author(s)

Bojing Jiang

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

Primary Investigator(s)

FZ

Fuzhong Zhang

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

Introduction: Mitsugumin 53 (MG53), typically expressed in skeletal and cardiac muscles of humans, rats and mice, plays an important role in muscle repair and regeneration after injury. It can repair muscles because it is an essential component of cell membrane repair machinery. Unfortunately, the therapeutic efficacy of MG53 after direct injection is very low due to its short half-life in blood circulation. To enhance the retention time of MG53 at specific injury sites, our strategy is to engineer MG53 by genetically fusing it with specific targeting cell peptides, including the ischemic myocardial targeting peptide (IMTP) and the cardiac targeting peptide (CTP). The recombination of MG53 and the cell targeting peptide would be used for therapies and drug delivery applications that aid in tissue repair and regeneration.

Materials and

Methods: E. coli cells were used to inoculate a seed culture of LB media, it was cultured at 37 °C with orbital shaking. This seed culture was used to inoculate the LB media in 2L orbital shaking flask at 37 °C until the optical density reached 0.6-0.8. All cultures were induced with 1 mL of ITPG and were allowed to grow orbital shaking at 16 °C for an additional 4 h. Cell pellets were collected by centrifugation and stored at - 80 °C until purification began.

Cell pellets were lysed in 30L of PBS, using sonication followed by centrifugation. The supernatant was loaded into Ni-NTA column, washed with PBS buffer containing 0 mM, 20 mM and 50 mM of imidazole. The supernatant was eluted with PBS buffer containing 300 mM imidazole. The purified proteins were dialyzed with PBS, lyophilized, and stored at –80 °C.

For SDS-PAGE gels, elution samples were resuspended in Laemmli sample buffer and incubated at 100 °C for 10 min. Electrophoresis was executed in Mini-PROTEAN Tetra Cells in Tris-glycine SDS buffer. For staining, all SDS-PAGE gels were submerged in a Coomassie Brillant Blue staining buffer then boiled for 1 min and placed on orbital shakers for 3 min at room temperature. For destaining, the gels were submerged in water and boiled for 10 min. Gel Images were acquired using Azure Biosystems cSeries.

Results, Conclusions, and Discussions: The SDS-PAGE gel images acquired from the elution samples were analyzed and labeled (Figure 2). The bands of each elution sample were compared to a protein standard marker to estimate the molecular weight. The estimated molecular weight from the SDS-PAGE gels and the expected molecular weight of each protein were compared to identify the target protein band (Table 2). The intensity of the band indicates the protein expression and concentration. By analyzing the target protein band, we were able to determine the expression of MG53-IMTP, MG53-CTP, and SpyCat-IMTP. The elution samples with the most intense bands were collected, which is shown by arrows in figure 2. From the collected elution samples, we were able to acquire a total yield of 7.5 mg for MG53-IMTP and 8.8 mg for MG53-CTP, each with a concentration of about 2 mg/mL (Table 1).

Our aim was to produce and purify recombinant proteins using synthetic biology techniques, to determine whether the intended protein is expressed. The results indicate success in the production and purification of MG53 recombinant proteins, MG53-IMTP and MG53-CTP. Furthering this study with additional testing for degradation and cell targeting properties in rats or mice could provide advancements in using the proteins for regenerative therapies and drug delivery applications.

Acknowledgements (Optional): Acknowledgements:

We would like to acknowledge the support of CEMB & SMARC at Washington University at St. Louis.

References:

Cai, C., Masumiya, H., Weisleder, N. et al. MG53 nucleates assembly of cell membrane repair machinery. Nat Cell Biol 11, 56–64 (2009). https://doi.org/10.1038/ncb1812

Wang, X., Chen, Y., Zhao, Z., Meng, Q., Yu, Y., Sun, J., ... & Shen, Z. (2018). Engineered exosomes with ischemic myocardium‐targeting peptide for targeted therapy in myocardial infarction. Journal of the American Heart Association, 7(15), e008737.

Li, Z.; Wang, L.; Yue, H.; Whitson, B.A.; Haggard, E.; Xu, X.; Ma, J. MG53, A Tissue Repair Protein with Broad Applications in Regenerative Medicine. Cells 2021, 10, 122. https://doi.org/10.3390/cells10010122

Li, J., Jiang, B., Chang, X., Yu, H., Han, Y., & Zhang, F. (2023). Bi-terminal fusion of intrinsically-disordered mussel foot protein fragments boosts mechanical strength for protein fibers. Nature communications, 14(1), 2127.