Week 09 HW -cell-free-systems

‘week-09-hw-cell-free-systems’

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Homework: Cell Free Systems

Homework Part A: General and Lecturer-Specific Questions

General homework questions

1. Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. 　　
Name at least two cases where cell-free expression is more beneficial than cell production.

従来の in vivo（生細胞内）法と比べて、cell-free protein synthesis（無細胞タンパク質合成）の主な利点を説明しなさい。  
特に、柔軟性と実験条件の制御という観点から述べなさい。  
また、細胞内での生産より無細胞発現のほうが有利な例を少なくとも2つ挙げなさい。

Cell-free protein synthesis
https://en.wikipedia.org/wiki/Cell-free_protein_synthesis#:~:text=CFPS has many advantages over,required for such a reaction.

Traditional protein expression methods present multiple challenges
https://www.biocompare.com/Editorial-Articles/594727-Advantages-of-Cell-Free-Protein-Expression/

A User’s Guide to Cell-Free Protein Synthesis
https://pmc.ncbi.nlm.nih.gov/articles/PMC6481089

Main advantages of cell-free protein synthesis　　無細胞タンパク質合成の主な利点 Faster 速い
A cell-free reaction can often be completed within 1–2 days, so results can be obtained much more quickly than with in vivo expression.
無細胞反応は 1〜2 日で完了することが多く、in vivo 発現よりも短時間で結果を得られる。

Greater flexibility and control 柔軟性と制御性が高い
Because CFPS is an open system, components such as DNA concentration, salts, cofactors, and engineered tRNAs can be directly adjusted.
CFPS は開放系であるため、DNA 濃度、塩濃度、補因子、改変 tRNA などを直接調整できる。

Less concern about toxicity 毒性の影響を受けにくい
The target protein does not need to be produced inside living cells, so toxic proteins are easier to handle.
標的タンパク質を生きた細胞内で作る必要がないため、毒性タンパク質でも扱いやすい。

Useful for toxic proteins 毒性タンパク質の発現に有利
Proteins that would damage or kill host cells during in vivo expression can often be produced more easily in a cell-free system.
細胞内で作ると宿主にダメージを与えるタンパク質でも、無細胞系では発現しやすい。

Useful for proteins containing unnatural amino acids 非天然アミノ酸を含むタンパク質の生産に有利
Since the reaction is open, modified tRNAs and unnatural amino acids can be introduced more easily.
開放系なので、改変 tRNA や非天然アミノ酸を導入しやすい。

Advantageous for membrane proteins 膜タンパク質の発現にも有利
Membrane proteins, which are often difficult to express correctly in living cells, can be tested under more controllable conditions in CFPS.
生細胞内では正しく発現させにくい膜タンパク質でも、条件を調整しながら発現を試せる。

2. Describe the main components of a cell-free expression system and explain the role of each component.    
無細胞発現系の主な構成要素を説明し、それぞれの役割を述べなさい。

https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2019.00248/full

Main components of a cell-free expression system　
無細胞発現系の主な構成要素

Comparison of the workflows of an in vivo system and a CFPS system
in vivo system と CFPS system の流れの比較図

A cell-free expression system consists of a DNA template, transcription and translation machinery, amino acids, nucleotides, an energy regeneration system, and cofactors, salts, and buffers.
These components respectively serve as the genetic blueprint, the synthesis machinery, the building materials, the energy supply, and the reaction environment.
無細胞発現系は、DNA テンプレート、転写・翻訳装置、アミノ酸、ヌクレオチド、エネルギー再生系、補因子・塩・バッファーからできており、
それぞれが「設計図」「合成機械」「材料」「エネルギー」「反応環境」となっている。

3. Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.   
なぜ無細胞系ではエネルギー供給・再生が重要なのか説明しなさい。  
また、あなたの無細胞実験で ATP を継続的に供給するために使える方法を1つ述べなさい。

Energy regeneration is critical in cell-free systems because transcription and translation consume large amounts of ATP and GTP.
Without continuous energy supply, these molecules are rapidly depleted, the reaction stops, and protein yield decreases.
One method to ensure continuous ATP supply is to use phosphoenolpyruvate (PEP) as an energy substrate.
PEP can regenerate ATP from ADP through pyruvate kinase, making it a common energy regeneration strategy in cell-free expression systems.

無細胞系では、転写と翻訳の両方に大量の ATP や GTP が必要であり、これらは反応中にすぐ消費されるため。
エネルギー再生がないと反応は短時間で止まり、目的タンパク質の収量も低下する。
ATP を継続的に供給する方法としては、phosphoenolpyruvate（PEP）を用いる方法がある。
PEP は pyruvate kinase を介して ADP から ATP を再生できるため、無細胞発現系のエネルギー供給法としてよく利用される。

4. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.   
原核生物由来と真核生物由来の無細胞発現系を比較しなさい。  
それぞれの系で作るタンパク質を1つずつ選び、なぜその系を選ぶのか説明しなさい。

Development of a robust Escherichia coli-based cell-free protein synthesis application platform
https://pmc.ncbi.nlm.nih.gov/articles/PMC7568173

Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems
https://pmc.ncbi.nlm.nih.gov/articles/PMC4676933

Prokaryotic cell-free expression system 原核生物由来の無細胞発現系 （for example, an E. coli extract 抽出液）　　

Characteristics 　　　 It is fast, easy to handle, and suitable for producing simple soluble proteins.
E. coli-based systems have been developed as platforms that can achieve high expression levels, and the experimental setup is relatively simple.
On the other hand, they are not well suited for complex eukaryotic post-translational modifications.

速くて扱いやすく、単純な可溶性タンパク質を作るのに向いている。
E. coli ベースの系では高い発現量が得られるプラットフォームが開発されており、実験系も比較的シンプル。
一方、複雑な真核生物型の翻訳後修飾は苦手。

Example protein
I would choose to produce sfGFP (superfolder GFP). sfGFP is soluble, easy to handle, and does not require complex glycosylation,
so it can be expressed efficiently in an E. coli-based system.
In fact, sfGFP is commonly used as a model protein in E. coli-based cell-free expression systems.
sfGFP（superfolder GFP）を作る。
sfGFP は可溶性で扱いやすく、複雑な糖鎖修飾を必要としないので、E. coli 系で十分に発現しやすい。
実際に、E. coli ベースの cell-free 系では sfGFP がよくモデルタンパク質として使われている。

Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives
https://pmc.ncbi.nlm.nih.gov/articles/PMC5042383

Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates
https://pmc.ncbi.nlm.nih.gov/articles/PMC5607253

Production of G protein‐coupled receptors in an insect‐based cell‐free system
https://pmc.ncbi.nlm.nih.gov/articles/PMC5599999

Characterisation of a cell-free synthesised G-protein coupled receptor https://pmc.ncbi.nlm.nih.gov/articles/PMC5430785

Eukaryotic cell-free expression system　真核生物由来の無細胞発現系
（for example, systems derived from CHO cells or insect cells (CHO や昆虫細胞由来）)

Characteristics：
It is well suited for producing complex eukaryotic proteins and membrane proteins.
For example, CHO-based cell-free systems contain ER-derived microsomal structures, which can support membrane insertion and some post-translational processes.
Insect cell-derived cell-free systems have also been used to synthesize membrane proteins such as GPCRs.

複雑な真核タンパク質や膜タンパク質に向いている。
たとえば CHO 由来の無細胞系には ER 由来の microsomal structures が含まれており、膜への挿入や一部の翻訳後過程を助けることができる。
昆虫細胞由来の無細胞系でも、GPCR のような膜タンパク質の合成が行われている。

Example proteins
I would choose to produce complex proteins such as GPCRs (G protein-coupled receptors) or antibodies.
These proteins require an appropriate membrane environment, correct folding, and often more complex assembly or modification, so eukaryotic cell-free systems are more suitable than E. coli-based systems.
Functional antibody production has been reported in CHO-based systems, and GPCR synthesis has been reported in insect cell-based systems.

GPCR（G protein-coupled receptor）や抗体のような複雑なタンパク質を作る。
こうしたタンパク質は、膜環境、正しい折りたたみ、複雑な会合や修飾が重要で、E. coli 系よりも真核由来の無細胞系のほうが適している。
CHO系では機能的な抗体、昆虫系では GPCR の合成例が報告されている。

5. How would you design a cell-free experiment to optimize the expression of a membrane protein? 
Discuss the challenges and how you would address them in your setup.    
膜タンパク質の発現を最適化するために、どのように無細胞実験を設計するか説明しなさい。   
また、想定される課題と、それに対して自分ならどう対処するかについて述べなさい。

High-throughput Cell-free Screening of Eukaryotic Membrane Protein Expression by R. Bruni, Q. Liu https://www.osti.gov/servlets/purl/1837204　　　

A cell-free system for functional studies of small membrane proteins
https://www.sciencedirect.com/science/article/pii/S0021925824023524

6. Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each.   
無細胞系で目的タンパク質の収量が低いと観察されたと仮定しなさい。   
考えられる理由を 3つ挙げ、それぞれについて トラブルシューティングの方法を提案しなさい。