Introduction
Cell free protein synthesis (CFPS) is a method for producing recombinant proteins without using live cells. This technology facilitates advancements in protein expression, metabolic engineering, therapeutic development, and education (Gregorio et al.). The foundational concept of CFPS, introduced by Eduard Buchner, involved converting sugar to ethanol and carbon dioxide using yeast extract. CFPS, also known as in-vitro translation, cell-free protein expression, or cell-free translation, is designed to meet the growing demand for complex proteins and drug lead molecules efficiently (Eric D. Carlson). Today, CFPS systems have generated numerous proteins with therapeutic significance.
CFPS systems are categorized based on the complexity of the cell extracts used: prokaryotic and eukaryotic. Prokaryotic CFPS systems utilize extracts from simpler organisms like Escherichia coli (Marshall et al., 2017). These systems are often employed in drug discovery due to their simpler structure and extensive research literature. However, prokaryotic systems lack the post-translational machinery required for producing complex mammalian proteins, leading to the development of eukaryotic CFPS systems. Examples include wheat germ lysates (WGL), cultured human cells like HeLa and K562, and Chinese Hamster Ovary (CHO) cells (Dondapati et al., 2020).
Historical Background
CFPS technology was first introduced between 1959 and 1962 through the Synthetic RNA and Poly-U experiments conducted by Nirenberg and Matthaei. In 1961, Nirenberg and Matthaei successfully synthesized a protein composed entirely of phenylalanine using artificial RNA with uracil nucleotides and E. coli extracts, without the need for live cells. This groundbreaking experiment led to the development of the CFPS platform. In the 1960s, CFPS was further utilized to explore the regulatory mechanisms of Escherichia coli lactose (Dopp et al., 2019).
Over the past 60 years, CFPS systems have gained popularity due to their efficiency, ease of preparation, and potential applications. CFPS platforms are categorized into high-adoption and low-adoption systems. High-adoption examples include E. coli, CHO cells, rabbit reticulocyte lysate, wheat germ, and HeLa cells. Low-adoption platforms include Streptomyces and Pseudomonas putida (Gregorio et al.). An example of a commercially available CFPS product is the PURExpress In Vitro Protein Synthesis Kit by New England Biolabs, USA.
Reasons for Popularity
CFPS platforms are favored for their cost-effectiveness compared to cell-based protein systems. The main steps in CFPS, illustrated in Figure 1, involve growing and harvesting cell lines, lysing the cells using sonication, and removing cell debris. The lysates, which can be stored for extended periods without losing functionality, are then used in protein synthesis by adding the necessary DNA templates and cofactors and allowing for incubation ranging from one to several hours (Khambhati et al., 2019).
Figure 1: Main steps involved in CFPS (Gregorio, N. E. et al 2019)
Compared to cell-based systems, CFPS offers faster protein production as it eliminates the need for cell transfection or gene cloning before cell growth and does not face cell viability constraints. In contrast, cell-based systems are generally slower and more time-consuming.
Advantages and Disadvantages of CFPS
CFPS platforms are highly valued in the pharmaceutical industry due to their open nature and rapid production times. Key applications include:
Production of Large and Toxic Proteins: CFPS systems can handle the synthesis of complex pharmaceutical-grade proteins. Eukaryotic lysates such as wheat germ and CHO cells are used for producing biologics with FDA approval, as they can perform human-compatible post-translational modifications (Gregorio et al., 2019
G-Protein Coupled Receptors and Drug Discovery: CFPS is ideal for synthesizing membrane proteins like ion channels and G-Protein coupled receptors, which are challenging to express in traditional cell-based systems due to their hydrophobic nature and potential cytotoxicity. CFPS also facilitates high-throughput ligand screening for drug development (Zemella et al., 2019).
CRISPR-Related Biomolecules: CFPS platforms, as characterized by the Noireaux Lab, have been used to create gene circuits and study complex diseases by synthesizing CRISPR-related nucleases and guide RNAs (Marshall et al., 2018).
Antibody Production: While cell-based systems are established for antibody production, CFPS platforms offer a faster and more cost-effective alternative. Examples include the production of IgGs in E. coli CFPS and Diphtheria toxoid antigen variants (DT5 and DT6).
Educational Use: CFPS systems are employed in academic settings to provide hands-on learning experiences. BioBits Kits, for instance, are used in classrooms for experiments such as producing fluorescent proteins and identifying fruit DNA (Stark et al., 2018).
The primary limitation of CFPS is its current impracticality for large-scale protein expression, however, there have been successful cases such as producing 70 kg of GMP-grade rhGM-CSF (Recombinant human granulocyte colony-stimulating factor) in 10 hours using Cell-Free Platform.
Conclusion
CFPS technology provides a versatile and efficient method for producing a range of proteins, from complex transmembrane proteins to simple ligands. Its ability to produce large quantities of proteins quickly and using various cell-free lysates can overcome the limitations of traditional cell-based synthesis, aiding in the treatment of rare diseases and advancing drug development. The growing adoption of CFPS across various applications underscores its potential in research, industrial production, and the development of novel drug lead molecules for addressing challenging diseases such as cancer, AIDS, and neurological disorders.
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