Method Optimization For Recombinant Protein Production

Research output: Contribution to conferencePosterpeer-review

Abstract

Introduction: Recombinant proteins are highly desirable for treating many blood coagulation disorders. They pose fewer risks than plasma-derived proteins. Production of recombinant proteins in mammalian cells (HEK293) is a delicate and lengthy process.

Aim: To optimize cell culture techniques and increase the yield of recombinantly prepared proteins.

Method: The optimized methods for protein expression, purification, and characterization involved several steps including protein transfection, stable cell line generation, purification using Q-Sepharose, and utilization of an FPLC machine. The transfection technique involved introducing the target plasmids into the cells using various transfection methods such as lipofection. The transfection process ensured that the desired proteins were expressed in the cells for subsequent analysis. The generation of a stable cell line ensured consistent expression of the target proteins, providing a reliable source for subsequent experiments. The ion exchange technique allowed for the separation and purification of the target proteins from the conditioned media, reducing background interference and enriching the protein of interest. The Q-Sepharose column, through its ion exchange properties, selectively bound the target proteins while allowing contaminants to flow through. The FPLC machine utilized a size-exclusion chromatography technique, which enabled the resolution of complex protein mixtures and the detection of target proteins with high sensitivity.

Result: In the current study and utilizing optimized methods, we purified and characterized several novel proteins and variants including VWF, ADAMTS13, ADAMTS7, and variants (D4, 3AE, 3A, D4-CK, FQ, Q2). The protein transfection technique allowed for the efficient delivery of target proteins into the cells, while the creation of stable cell lines ensured their consistent expression. Additionally, the purification process using a Q-Sepharose column enabled the isolation of the desired proteins with high specificity. Lastly, the FPLC machine facilitated the separation and analysis of the purified proteins.

Conclusion: Several novel coagulation proteins were prepared and characterized using optimized methods. They will be used for subsequent experiments.
Original languageAmerican English
Pages49
StatePublished - 16 Feb 2024
Event
Oklahoma State University Center for Health Sciences Research Week 2024
- Oklahoma State University Center for Health Sciences, Tulsa, United States
Duration: 13 Feb 202417 Feb 2024
https://medicine.okstate.edu/research/research_days.html

Conference

Conference
Oklahoma State University Center for Health Sciences Research Week 2024
Country/TerritoryUnited States
CityTulsa
Period13/02/2417/02/24
Internet address

Keywords

  • VWF
  • ADAMTS7
  • ADAMTS13
  • transfection
  • stable cellline
  • recombinant

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