preparation of supplemented leishmania translational extract from cell concentrate

<meta charset="utf8"></head><body>Leishmania Translasyonel Ekstrakt (LTE) Üretimi için Kolaylaştırılmış Protokol

In the 1960s, cell-free protein expression systems played a pivotal role in uncovering the genetic code1. However, prokaryotic cell-free protein expression systems, mainly based on E. coli, currently dominate both laboratory and commercial applications. While E. coli-based systems offer advantages such as cost-effectiveness, scalability, and high expression yields, they face challenges when producing multi-domain proteins in their active forms and facilitating the assembly of protein complexes2,3. In the present day, commonly used forms of eukaryotic Cell-Free Protein Synthesis (CFPS) include wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and insect cell lysate (ICL)4,5,6. This work introduces an alternative eukaryotic cell-free system that is both straightforward and scalable, based on the unicellular flagellate parasite Leishmania tarentolae.
Leishmania tarentolae can be cultivated easily in flasks using cost-effective media and can also be scaled up in bioreactors to achieve higher cell density. The presence of endogenous mRNAs in cell lysate, which might otherwise compete with introduced messages, can be neutralized using antisense oligonucleotides targeting the conserved Leishmania mRNA splice leader sequence7. Unlike its close relative Leishmania major, which causes human disease, L. tarentolae infects the moorish gecko (Tarentolae mauritanica), making it suitable for cultivation in PC2 laboratory environments without the need for special precautions. It has previously been used as a transgenic organism for in vivo protein expression8.
To facilitate template priming in cell-free systems, universal sequences have been designed based on polymeric RNA structures that enhance translational initiation9. These species-independent translation sequences (SITS) are applicable to both prokaryotic and eukaryotic cell-free systems and are suitable for introducing genetic information into LTE. While this protocol does not provide a detailed explanation of vector construction for LTE cell-free protein expression, optimization and quality control require suitable vectors containing fluorophore fusions of the desired proteins of interest downstream of the SITS site. For this purpose, appropriate LTE vectors have been deposited with the Addgene gene repository, such as the pCellFree_G03 vector, which encodes an N-terminal eGFP fusion to the desired protein of interest using Gateway cloning sites.
LTE has proven its value in a wide range of applications requiring protein expression, including the analysis of protein self-assembly10,16, production of human integral membrane proteins17, research on antiviral drug candidates18, the development of biotechnologically useful enzymes19, prototyping protein biosensors20,21, and the study of biologics from hookworms22. LTE has also been instrumental in mapping Protein-Protein interaction networks in the fields of virology and cellular structures21,32. LTE has been benchmarked to perform similarly to other eukaryotic cell-free systems in expressing full-length, monodispersed, and non-aggregated proteins33, all while offering more cost-effective and scalable production.
This protocol provides techniques for cultivating and disrupting the host organism, preparing lysate, and supplementing a feeding solution (FS) for coupled transcription/translation protein expression. Additionally, it includes a protocol for optimizing production batches. In the initial version of the Leishmania cell-free system, undesired batch-to-batch variation was observed in expression levels, the fraction of full-length proteins, and the presence of protein aggregates, leading to the disposal of batches34. Subsequent protocol improvements were made to address this issue25. The current protocol builds upon these improvements, allowing individual batches to be optimized for peak protein expression and size. It achieves this by closely controlling cell-disrupter loading (measured as optical density at 600 nm; OD600nm) and normalizing the resulting lysate output using absorbance at 280 nm (Abs280nm). Furthermore, it incorporates a method for partially supplementing the lysate with rNTP and magnesium during manufacturing, with subsequent optimization of these feed solution components during test expressions. Although this optimization is presented as an option in the protocol, it is strongly recommended by the authors.

<meta charset="utf8"></head><body>Yazar Spotlight: LTE Ökaryotik Hücresiz Protein Ekspresyon Sistemini Kullanarak Protein Üretimi

Rekombinant Protein Üretimi için Leishmania Tarentolae Türetilmiş Hücresiz Protein Ekspresyon Sistemi olan LTE'nin Üretimi ve Optimizasyonu

Our cell-free protein expression system, which typically requires a cold chain for transport, now has a pioneering freeze-dried version. This version remains stable at ambient temperatures, making it the first eukaryotic system available in this format. This simplifies transportation to labs and field applications.The current experimental challenges of cell-free protein production include optimizing reaction conditions to ensure that recombinant proteins are properly folded and active. Although the cost-effectiveness of cell-free techniques versus conventional in vivo protein expression remains a significant challenge, the simplicity of our eukaryotic LTE cell-free system to produce helps alleviate this. The LTE cell-free system facilitates swift reconstitution and analysis of protein networks and biosynthetic pathways.Its integration with technologies like AlphaLISA and in vivo assays enables functional analysis of complex systems, including the protein interaction network of SARS-CoV-2 and the hookworm secretome. The LTE cell-free system enables rapid, cost-effective production of hard-to-express proteins in a multiplex format. It offers easy optimization for protein synthesis, excelling in producing complex or toxic proteins.The system is scalable and provides consistent batch-to-batch quality.

Hücre Konsantresinden Takviye Edilmiş Leishmania Translasyonel Ekstraktının Hazırlanması

preparation-supplemented-leishmania-translational-extract-from-cell

Research

JoVE Journal

Bioengineering

Preparation of Supplemented Leishmania Translational Extract from Cell Concentrate

To begin, harvest the overnight cultures of Leishmania tarentolae. Measure the harvest optical density of the cultures at 600 nanometers, using a 1:10 dilution in culture medium. Calculate the target concentrate volume to achieve a final optical density of 300 at 600 nanometers.Transfer the harvested cell culture into suitable centrifuge bottles. Centrifuge the bottles at 2, 500 G for 10 minutes at four degrees Celsius. Then carefully decant the supernatant into a receptacle for culture waste.Wash the cell pellet in 500, 200, and 20 milliliters of SEB buffer. Then pour the SEB resuspended concentrate into a suitable washed volumetric cylinder. Make up the volume to the target volume using cold SEB and then gently mix using a pipette.To lyse the cell concentrate, transfer it into a pre-cooled nitrogen cavitation device. Pressurize to 70 bars and incubate for 45 minutes on ice. Next, open the vent on the cavitation device and expel the lysate into a cleaned vacuum receiver flask placed on ice.Then transfer the lysate into centrifuge tubes and centrifuge at 10, 000 G for 15 minutes at four degrees Celsius. After transferring the supernatant to fresh tubes, centrifuge the lysate at 30, 000 G for 15 minutes at four degrees Celsius. Then transfer the supernatant to a fresh centrifuge tube placed on ice.Set up four PD-10 gravity-fed gel filtration columns per 10 milliliters of lysate in a rack format that allows them to drip into a suitable collection tray. Add 2.5 milliliters of lysate into each column. Once it passes into the column, add 0.5 milliliters of elution buffer to settle the lysate.Remove the waste tray and place a fresh collection tray beneath the columns. Then add 2.5 milliliters of elution buffer to elute the gel-filtered lysate. Collect the gel-filtered lysate in a fresh centrifuge tube.Next, measure the absorbance of the filtered lysate at 280 nanometers with a NanoDrop spectrophotometer. Add five times feeding solution to the lysate in a 2:5 ratio. Then vortex the mixture thoroughly.Aliquot the mixture into 1.5 milliliter microcentrifuge tubes and then snap-freeze the tubes in liquid nitrogen for future use.