cell-free-expression

Name: cell-free-expression
Author: adaptyvbio

41installs20forks

How to install cell-free-expression?

adaptyvbio/protein-design-skills/cell-free-expression

$npx -y skills add adaptyvbio/protein-design-skills --skill cell-free-expression

Installs into the current project.

Summary

Guidance for cell-free protein synthesis (CFPS) optimization. Use when: (1) Planning CFPS experiments, (2) Troubleshooting low yield or aggregation, (3) Optimizing DNA template design for CFPS, (4) Expressing difficult proteins (disulfide-rich, toxic, membrane).

Files · 1

View on GitHub

SKILL.md

1	# Cell-Free Protein Synthesis (CFPS)
2
3	## System Selection Guide
4
5	\| System \| Best For \| Yield \| PTMs \| Disulfides \| Cost \|
6	\|--------\|----------\|-------\|------\|------------\|------\|
7	\| E. coli extract \| Rapid prototyping, prokaryotic proteins \| High (100-400 μg/mL) \| None \| Poor (reducing) \| Low \|
8	\| E. coli PURE \| Defined conditions, unnatural AAs \| Medium (50-150 μg/mL) \| None \| Controllable \| High \|
9	\| Wheat germ \| Eukaryotic proteins, membrane proteins \| High (100-500 μg/mL) \| Limited \| Moderate \| Medium \|
10	\| Rabbit reticulocyte \| Mammalian proteins, post-translational studies \| Low (10-50 μg/mL) \| Some \| Poor \| High \|
11	\| Insect (Sf21) \| Glycoproteins, complex folds \| Medium (50-100 μg/mL) \| Glycosylation \| Good \| High \|
12	\| HeLa/CHO \| Native mammalian proteins \| Low (10-50 μg/mL) \| Full mammalian \| Good \| Very High \|
13
14	---
15
16	## CFPS Troubleshooting Matrix
17
18	\| Problem \| Likely Causes \| Design Fix \| Reagent Fix \|
19	\|---------\|---------------\|------------\|-------------\|
20	\| No expression \| Rare codons at N-terminus, poor RBS \| Codon optimize first 30 codons \| Use BL21-CodonPlus extract \|
21	\| Low yield \| Strong mRNA secondary structure, template issues \| Optimize 5' UTR (ΔG > -5 kcal/mol) \| Increase Mg²⁺ (10-18 mM), ATP \|
22	\| Aggregation \| Hydrophobic protein, fast translation \| Add solubility tags (MBP, SUMO) \| Add 0.1% Tween-20, chaperones \|
23	\| Inactive protein \| Misfolding, missing cofactors \| Slow translation (use rare codons!) \| Add GroEL/ES, DnaK/J \|
24	\| Truncation \| Rare codon clusters, mRNA instability \| Remove AGG/AGA/CUA clusters \| Supplement rare tRNAs \|
25	\| Degradation \| Proteolysis \| N-terminal Met-Ala \| Add protease inhibitors \|
26
27	---
28
29	## Codon Optimization for CFPS
30
31	### Codons to Avoid in E. coli CFPS
32
33	\| Codon \| Amino Acid \| Issue \| tRNA Abundance \|
34	\|-------\|------------\|-------\|----------------\|
35	\| AGG \| Arg \| Very rare, stalling \| 0.2% \|
36	\| AGA \| Arg \| Very rare, stalling \| 0.4% \|
37	\| CUA \| Leu \| Low abundance \| 0.4% \|
38	\| AUA \| Ile \| Rare \| 0.5% \|
39	\| CGA \| Arg \| Inefficient decoding \| 0.6% \|
40	\| CCC \| Pro \| Can cause pausing \| 0.5% \|
41	\| GGA \| Gly \| Moderate \| 1.1% \|
42
43	### Design Rules
44
45	1. First 30 codons: Most critical - use only high-frequency codons
46	2. Rare codon clusters: Avoid 2+ rare codons within 10 nt
47	3. Rare codon content: Keep overall <5% of coding sequence
48	4. GC content: Target 40-60% for balanced expression
49	5. Avoid runs: No >6 consecutive G or C residues (secondary structure)
50	6. Strategic slow codons: Place rare codons between domains (aids folding!)
51
52	### When to Use Rare Codons
53	- Domain boundaries (allow cotranslational folding)
54	- Before complex structural elements
55	- When protein is prone to misfolding
56
57	---
58
59	## mRNA Template Design
60
61	### 5' UTR Optimization
62
63	\| Element \| Optimal Design \| Impact \|
64	\|---------\|----------------\|--------\|
65	\| RBS (SD sequence) \| AGGAGG, 7-9 nt from start \| Ribosome binding \|
66	\| Spacing \| 7 nt between SD and AUG \| Translation initiation \|
67	\| Secondary structure \| ΔG > -5 kcal/mol \| Accessibility \|
68	\| Upstream AUG \| Avoid (causes false starts) \| Reduces truncations \|
69
70	### Secondary Structure Targets
71
72	\| Region \| Ideal ΔG \| Impact \|
73	\|--------\|----------\|--------\|
74	\| -30 to +30 around AUG \| > -5 kcal/mol \| Translation initiation \|
75	\| Full 5' UTR \| > -10 kcal/mol \| Ribosome loading \|
76	\| RBS accessibility \| Unpaired \| Critical \|
77
78	### Template Format
79
80	\| Format \| Advantages \| Disadvantages \|
81	\|--------\|------------\|---------------\|
82	\| Plasmid \| Stable, high yield \| Requires cloning \|
83	\| Linear PCR \| Fast, no cloning \| May need stabilization \|
84	\| mRNA \| Direct translation \| Unstable, expensive \|
85
86	---
87
88	## Disulfide Bond Formation
89
90	### System Capabilities
91
92	\| System \| Native Disulfide Support \| Additives Needed \|
93	\|--------\|--------------------------\|------------------\|
94	\| Standard E. coli extract \| Poor (DTT present) \| IAM, PDI, GSSG/GSH \|
95	\| Oxidizing E. coli extract \| Good \| Pre-oxidized glutathione \|
96	\| Wheat germ \| Moderate \| Lower DTT, add PDI \|
97	\| PURE system \| Minimal \| Full oxidative system \|
98	\| Insect/Mammalian \| Good \| Microsome membranes \|
99
100	### Oxidative Folding Protocol (E. coli extract)
101
102	```
103	1. Deplete DTT from extract (dialysis or treatment with IAM 5 mM)
104	2. Add oxidized/reduced glutathione: 4 mM GSSG, 1 mM GSH (4:1 ratio)
105	3. Add 10 μM PDI (protein disulfide isomerase)
106	4. Optional: Add 5 μM DsbC (disulfide isomerase)
107	5. Express at 25°C (not 37°C) for better folding
108	6. Incubation time: 4-6 hours
109	```
110
111	### Disulfide-Rich Protein Tips
112	- Start with wheat germ or oxidizing extract
113	- Use PURE system for precise control
114	-

People who liked this also viewed

6 picks

Type

cell-free-expression

41installs20forks

View on GitHub

How to install cell-free-expression?

adaptyvbio/protein-design-skills/cell-free-expression

$npx -y skills add adaptyvbio/protein-design-skills --skill cell-free-expression

Installs into the current project.

Summary

Files · 1

View on GitHub

SKILL.md

1	# Cell-Free Protein Synthesis (CFPS)
2
3	## System Selection Guide
4
5	\| System \| Best For \| Yield \| PTMs \| Disulfides \| Cost \|
6	\|--------\|----------\|-------\|------\|------------\|------\|
7	\| E. coli extract \| Rapid prototyping, prokaryotic proteins \| High (100-400 μg/mL) \| None \| Poor (reducing) \| Low \|
8	\| E. coli PURE \| Defined conditions, unnatural AAs \| Medium (50-150 μg/mL) \| None \| Controllable \| High \|
9	\| Wheat germ \| Eukaryotic proteins, membrane proteins \| High (100-500 μg/mL) \| Limited \| Moderate \| Medium \|
10	\| Rabbit reticulocyte \| Mammalian proteins, post-translational studies \| Low (10-50 μg/mL) \| Some \| Poor \| High \|
11	\| Insect (Sf21) \| Glycoproteins, complex folds \| Medium (50-100 μg/mL) \| Glycosylation \| Good \| High \|
12	\| HeLa/CHO \| Native mammalian proteins \| Low (10-50 μg/mL) \| Full mammalian \| Good \| Very High \|
13
14	---
15
16	## CFPS Troubleshooting Matrix
17
18	\| Problem \| Likely Causes \| Design Fix \| Reagent Fix \|
19	\|---------\|---------------\|------------\|-------------\|
20	\| No expression \| Rare codons at N-terminus, poor RBS \| Codon optimize first 30 codons \| Use BL21-CodonPlus extract \|
21	\| Low yield \| Strong mRNA secondary structure, template issues \| Optimize 5' UTR (ΔG > -5 kcal/mol) \| Increase Mg²⁺ (10-18 mM), ATP \|
22	\| Aggregation \| Hydrophobic protein, fast translation \| Add solubility tags (MBP, SUMO) \| Add 0.1% Tween-20, chaperones \|
23	\| Inactive protein \| Misfolding, missing cofactors \| Slow translation (use rare codons!) \| Add GroEL/ES, DnaK/J \|
24	\| Truncation \| Rare codon clusters, mRNA instability \| Remove AGG/AGA/CUA clusters \| Supplement rare tRNAs \|
25	\| Degradation \| Proteolysis \| N-terminal Met-Ala \| Add protease inhibitors \|
26
27	---
28
29	## Codon Optimization for CFPS
30
31	### Codons to Avoid in E. coli CFPS
32
33	\| Codon \| Amino Acid \| Issue \| tRNA Abundance \|
34	\|-------\|------------\|-------\|----------------\|
35	\| AGG \| Arg \| Very rare, stalling \| 0.2% \|
36	\| AGA \| Arg \| Very rare, stalling \| 0.4% \|
37	\| CUA \| Leu \| Low abundance \| 0.4% \|
38	\| AUA \| Ile \| Rare \| 0.5% \|
39	\| CGA \| Arg \| Inefficient decoding \| 0.6% \|
40	\| CCC \| Pro \| Can cause pausing \| 0.5% \|
41	\| GGA \| Gly \| Moderate \| 1.1% \|
42
43	### Design Rules
44
45	1. First 30 codons: Most critical - use only high-frequency codons
46	2. Rare codon clusters: Avoid 2+ rare codons within 10 nt
47	3. Rare codon content: Keep overall <5% of coding sequence
48	4. GC content: Target 40-60% for balanced expression
49	5. Avoid runs: No >6 consecutive G or C residues (secondary structure)
50	6. Strategic slow codons: Place rare codons between domains (aids folding!)
51
52	### When to Use Rare Codons
53	- Domain boundaries (allow cotranslational folding)
54	- Before complex structural elements
55	- When protein is prone to misfolding
56
57	---
58
59	## mRNA Template Design
60
61	### 5' UTR Optimization
62
63	\| Element \| Optimal Design \| Impact \|
64	\|---------\|----------------\|--------\|
65	\| RBS (SD sequence) \| AGGAGG, 7-9 nt from start \| Ribosome binding \|
66	\| Spacing \| 7 nt between SD and AUG \| Translation initiation \|
67	\| Secondary structure \| ΔG > -5 kcal/mol \| Accessibility \|
68	\| Upstream AUG \| Avoid (causes false starts) \| Reduces truncations \|
69
70	### Secondary Structure Targets
71
72	\| Region \| Ideal ΔG \| Impact \|
73	\|--------\|----------\|--------\|
74	\| -30 to +30 around AUG \| > -5 kcal/mol \| Translation initiation \|
75	\| Full 5' UTR \| > -10 kcal/mol \| Ribosome loading \|
76	\| RBS accessibility \| Unpaired \| Critical \|
77
78	### Template Format
79
80	\| Format \| Advantages \| Disadvantages \|
81	\|--------\|------------\|---------------\|
82	\| Plasmid \| Stable, high yield \| Requires cloning \|
83	\| Linear PCR \| Fast, no cloning \| May need stabilization \|
84	\| mRNA \| Direct translation \| Unstable, expensive \|
85
86	---
87
88	## Disulfide Bond Formation
89
90	### System Capabilities
91
92	\| System \| Native Disulfide Support \| Additives Needed \|
93	\|--------\|--------------------------\|------------------\|
94	\| Standard E. coli extract \| Poor (DTT present) \| IAM, PDI, GSSG/GSH \|
95	\| Oxidizing E. coli extract \| Good \| Pre-oxidized glutathione \|
96	\| Wheat germ \| Moderate \| Lower DTT, add PDI \|
97	\| PURE system \| Minimal \| Full oxidative system \|
98	\| Insect/Mammalian \| Good \| Microsome membranes \|
99
100	### Oxidative Folding Protocol (E. coli extract)
101
102	```
103	1. Deplete DTT from extract (dialysis or treatment with IAM 5 mM)
104	2. Add oxidized/reduced glutathione: 4 mM GSSG, 1 mM GSH (4:1 ratio)
105	3. Add 10 μM PDI (protein disulfide isomerase)
106	4. Optional: Add 5 μM DsbC (disulfide isomerase)
107	5. Express at 25°C (not 37°C) for better folding
108	6. Incubation time: 4-6 hours
109	```
110
111	### Disulfide-Rich Protein Tips
112	- Start with wheat germ or oxidizing extract
113	- Use PURE system for precise control
114	-

People who liked this also viewed

6 picks

Type