Table 2.

Comparison of different cell-free systems reported in the literature with their significant properties

Cell-free system origin	Strengths	Weaknesses	Protein examples
Prokaryotic
Escherichia coli	CF lysates are simple to prepare and very efficient CFPS is simple with a high yield of proteins within a few hours (depends on protein) Suitable for establishment in large scale in many research laboratories and companies Cost effective and doesn’t need a large infrastructure Highly stable and receptive to external supplements Lysates used for point-of-care testing [30, 31] Wealth of genetic tools and literature	No PTMs Not suitable for MPs and proteins whose folding function depend on PTMs Lack of translationally active endogenous microsomes Need to supply additional membrane solubilization supplements for MPs Larger proteins (> 70 kDa) prone to higher aggregation or truncated products [47] Contamination with endotoxins	Trimeric influenza hemagglutinin stem domain (6-h batch): 400 μg/mL [48] NavSp1p (2-h batch): 20 μg/mL [49] PfFNT (CECF overnight): 4 mg/mL [50] TDH (2-h batch): 300 μg/mL [51] Kv 1.3 (4-h batch): 15–25 μg/mL [52] hVDAC1: 10 mg/mL [53]
Streptomyces	Suitable for GC-rich proteins [34] Simple and robust preparation of lysates	No PTMs reported No endogenous microsomes	EGFP (3-h batch): 50 µg/mL and (48-h CECF): 282 µg/mL [34] Tbr (P,Q,N,I) and TEII (3-h batch): 11–17 µg/mL [34]
Bacillus subtilis	Alternative to the E. coli-based system Wealth of genetic tools and literature	No PTMs Lack of translationally active endogenous microsomes Relatively very new and limited reports	GFP (2.5-h batch): 22 µg/mL [32] Luciferase (1-h batch): 40–150 μg/mL [54]
Vibrio natrigens	Generate a high volume of the active lysate (8–12 mL/1 L culture) [12] Robust lysate preparation and high metabolic efficiency Highly stable at room temperature for 1 wk Higher ribosomal concentration per cell compared with E. coli	Limited applications until now Relatively very new	Opistoporin 1: 278 µg/mL [12] Cecropin A: 22 µg/mL [12] Cecropin P1: 96.8 µg/mL [12] EGFP: 400 µg/mL [55]
Eukaryotic
Insect Spodoptera frugiperda 21 (Sf21)	Mimic the Sf21 cell-based production PTMs (N-glycosylation, disulfide bridging, and lipidation) Suitable for a wide range of eukaryotic and complex proteins Presence of translationally active endogenous microsomes [14] High yields in CECF mode Endotoxin free	Low yields especially in the batch mode Cost ineffective and difficult to establish unlike E. coli-based system	KcSA (4 h batch): 8 µg/mL [56] hEGFR (2-h batch): 6 µg/mL [14] (3 × 2-h batch [repetitive]): 15 µg/mL [14] (24-h CECF): 285 µg/mL [57]
Chinese hamster ovary (CHO)	Mimic the CHO cell-based production PTMs (N-glycosylation, disulfide bridging, and lipidation) Suitable for a wide range of eukaryotic and complex proteins Presence of translational active endogenous microsomes [45] High yields in CECF mode Endotoxin free Lysates used for point-of-care testing [30]	Low yields especially in the batch mode [58] Cost ineffective and difficult to establish unlike E. coli-based system	Streptokinase (CECF): 500 µg/mL [59] Human TLR9 receptor [18] (3-h batch): 21 µg/mL (48-h CECF): 900 µg/mL hEGFR [46] (batch): 40 µg/mL (CECF): 800 µg/mL
Wheat germ	No codon optimization necessary Highly efficient translation machinery, suitable for a wide variety of proteins including MPs and eukaryotic proteins that do not depend on PTMs for their functionality [47] Alternative to E. coli-based system for producing high yield of proteins Highly stable and resistant to external supplements Promising system for vaccine development No endogenous mRNA	Lack of endogenous microsomes Need to supply additional solubilization supplements for MPs CF lysate preparation is time consuming Lack of glycosylation	GFP (3-h batch): 1.6 mg/mL [43] hTERT (48-h CECF): 1.5 mg/mL [58] HRH1 (24-h CECF): 800 µg/mL [60]
Tobacco BY-2	Very fast lysate preparation (4–5 h) Presence of endogenous microsomes allowing PTMs (glycosylation and disulfide bond) High translational efficiency	Relatively undeveloped PTMs not well characterized	Vitronectin-specific full-size human antibody M12 (18-h batch): 150 µg/mL [35] Heparin-binding EGF-like growth factor (HbEGF) (18-h batch): 25 µg/mL [35] Glucose oxidase: 7.34 U/mL [35] hCNTF (16-h batch): 20 µg/mL [58, 61]
Cultured human cells (HeLa, K562 extracts)	Mimic the human cell-based production PTMs (N-glycosylation, disulfide bridging, and lipidation) Suitable for a wide range of eukaryotic and complex proteins [47] Presence of translationally active endogenous microsomes Endotoxin free	Low yields and not well established Cost ineffective and difficult to establish unlike E. coli-based system	Luciferase (2-h batch): 21 µg/mL [62] GST (CECF HeLa): 50 µg/mL [63] hCNTF (6-h batch): 50 µg/mL [61]
Rabbit reticulocyte	Cap-independent translation PTMs Suitable for large complex proteins [64] Easy to prepare	Low translation efficiency Difficult to prepare lysates Need to externally supply exogenous microsomes for protein folding Ethical issues	nAChR: no data [44] HBc: no data [65] EGFP (batch): 30 µg/mL [66] FhSAP2 (1.5-h batch): 500 µg/mL [67]
Leishmania tarentolae	Lowest aggregation propensity Better solubility of expressed proteins Background translation of endogenous mRNA suppressed Ease of handling similar to E. coli and offer complete eukaryotic expression	Protein synthesis only reported for GFP Not so well established No PTMs reported	EGFP (batch): 300 µg/mL [66] NPT4 (16-h CECF): 50 µg/mL [68]
Neurospora	Rapid and inexpensive	No report of endogenous microsomes No report of PTMs established	Luciferase (30-min batch): 2.5 µg/mL [37]
Saccharomyces cerevisiae	Easy to prepare Wealth of genetic tools available and cell culture literature Contains eukaryotic folding machinery Lack of endotoxins	No PTMs reported Low batch yields CF system underdeveloped	HPV 58 L1 (3-h batch): 60 µg/mL [69] CAT (5-h batch): 10 µg/mL [38] EPO (6-h CECF): 40 µg/mL [70]

CAT chloramphenicol acetyl transferase, CECF continuous exchange cell-free synthesis, CF cell-free, CFPS cell-free protein synthesis, E. coli Escherichia coli, EGFP enhanced green fluorescent protein, EPO human erythropoietin, FhSAP2 Fasciola hepatica saposin-like protein-2, GC guanine-cytosine, GFP green fluorescent protein, GST glutathione S-transferase, HBc hepatitis B-core protein, hCNTF human ciliary neurotrophic factor, hEGFR human epidermal growth factor receptor, HPV 58 human papillomavirus-58, HRH1 human histamine H1 Receptor, hTERT human telomerase reverse transcriptase, hVDAC1 human voltage dependent anionic channel, KcSA pH-gated potassium channel, Kv1.3 voltage gated potassium channel, MPs membrane proteins, NavSp1p Silicibacter pomeroyi voltage-gated sodium channel, nAChR nicotinic acetylcholine receptor, NPT4 human sodium phosphate transporter 4, PfFNT plasmodium lactate transporter, PTM post-translational modification, TDH thermostable direct hemolysin, Tbr (P,Q,N,I) genes involved in the biosynthesis of peptide tambromycin, TEII type II thioesterase, TLR9 toll-like receptor 9