Biology Reference
In-Depth Information
TABLE 15.1
Common Energy Sources and the Relative Cost of Cell-Free Protein
Production Using Commercial Kits
CFPS Type
Commonly used
Energy Sources
Extract Preparation Time Relative Cost (per
ng Protein Yield)
Phosphoenolpyruvate
146
Pyruvate
146
Acetyl kinase/acetyl
phosphate
147
Glucose
148
Creatine kinase/creatine
phosphate
15
Creatine kinase/creatine
phosphate/glucose
149
Creatine kinase/creatine
phosphate/cAMP
150
B
$0.701
75
Escherichia coli
lysate
12 days
3
4
wheat germ
lysate
Creatine kinase/creatine
phosphate
70
4
5 days
$1.50
B
9.25
Insect cell
lysate
Creatine kinase/creatine
phosphate
151
1
2 days
$4
B
$11
Purified
component
Creatine kinase/creatine
phosphate
152
Extract not used, 32
individually purified
components
B
100
75
Rabbit
reticulocyte
cell lysate
Creatine kinase/creatine
phosphate
153
4 days for rabbit treatment,
1 day for extract prep
$30
B
Cost assessed using maximum yields of respective commercial kits.
Expressway Maxi Cell-free E. coli Expression System from Invitrogen, S30 T7 High-yield Expression Kit from Promega.
Wheat Germ Extract Cell Free Protein Synthesis Kits from Genecopoeia.
TnT
s
T7 Insect Cell Extract Protein Expression System from Promega.
TnT-coupled Reticulocyte Lysate System from Promega.
PURExpress In vitro Protein Synthesis Kit from NEB.
285
major eukaryotic cell-free systems.
70,71
Using a cell-free protein-producing gel, one group
reported yields as high as 5 mg/ml, approaching the highest yields reported for
E. coli
-based
cell-free synthesis, but at much lower volumes.
71
Unfortunately, wheat germ extract
preparation is more costly and more labor-intensive than
E. coli
-based systems.
Rabbit reticulocyte-based cell-free systems require a highly technical and difficult
extract preparation method, but are used because of their ability to enable some post-
translational modifications.
72
74
Low expression yields linked to the significant amount
of ribonuclease M in the lysate, however, limit their utility.
75
Additionally, the collection
of sufficient amounts of reticulocytes requires harmful chemical treatments of rabbits.
73
Commonly produced from
Spodoptera frugiperda
cells, insect-based CFPS provide an effective
combination of ample protein yield and post-translational modifications.
76
Commercial
insect cell-based systems have reached yields of up to 50
g/ml.
76
They also have been used
successfully for several forms of post-translational modifications including glycosylation,
isoprenylation, acetylation, N-myristoylation, and others.
77
79
Due to the cost of insect
cell cultivation and extract preparation, insect cell-based cell-free systems are more costly
than
E. coli
-based or wheat germ-based systems. While each of the CFPS systems developed
to date has their merits and trade-offs, we turn our attention to several case studies that
highlight the advantages of having direct access and control to the reaction conditions, as
well as emerging applications made possible by recent technical advances.
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