Cell-free protein expression is performed without the use of living cells. Instead, all components needed to transcribe DNA to RNA and translate the RNA to protein (e.g. ribosomes, tRNAs, enzymes, cofactors, amino acids) are provided in solution for use in vitro. Generally, such solutions are obtained through making a cell lysate from a desired cell type. Cell-free mixtures have been made from both bacterial and eukaryotics cells.

1. Cell Free Protein
ExpressionSystem
Tasmina Ferdous Susmi

2. Cell-Free
Protein
Expression
 The production of recombinant proteins in solution using
bimolecular translation machinery extracted from cells.
 Performed without the use of living cells.
 All components needed to transcribe DNA to RNA and translate
the RNA to protein (e.g. ribosomes, tRNAs, enzymes, cofactors,
amino acids) are provided in solution for use in vitro.
 Such solutions are obtained through making a cell lysate from a
desired cell type.

3. Cell-Free
Protein
Expression
 Also known as in vitro translation, cell-free protein expression,
cell-free translation, or in vitro protein expression.
 Cell-free mixtures have been made from both bacterial and
eukaryotic cells.
 Cell-free systems are generally not practical for large-scale protein
expression.

4. Cell-Free
Protein
Expression
Mechanism
 Two basic components are needed to accomplish in vitro protein
expression:
(1) the genetic template (mRNA or DNA) encoding the target protein
and
(2) a reaction solution containing the necessary transcriptional and
translational molecular machinery.
 Cell extracts supply all or most of the molecules of the reaction
solution, including:
1. RNA polymerases for mRNA transcription
2. ribosomes for polypeptide translation
3. tRNA and amino acids
4. enzymatic cofactors and an energy source
5. cellular components essential for proper protein folding

5. Mechanism
Continued
 Cell lysates provide the correct composition and proportion of
enzymes and building blocks required for translation.
 Usually, an energy source and amino acids must also be added to
sustain synthesis.
 Cell membranes are removed to leave only the cytosolic and
organelle components of the cell (hence the term, “cell-free
extracts”).
 The first types of lysates developed for cell-free protein expression
were derived from prokaryotic organisms.
 More recently, systems based on extracts from insect cells,
mammalian cells and human cells have been developed and made
commercially available.

6. Selection of
Cell-Free
Protein
Expression

7. Types of cell-
free extracts
 Extracts used for cell-free protein expression are made from
systems known to support high level protein synthesis.
 The first known cell-free extracts capable of supporting translation
were made from E. coli.
 Extracts made from these eukaryotic systems contain all the
necessary cellular macromolecules like ribosomes, translation
factors and tRNAs required for efficient protein synthesis.
 These lysates are pretreated with micrococcal nuclease to support
translation of only an exogenously supplied message.
 Once endogenous genes and transcripts are removed, inhibitors
for the enzyme RNase are supplemented to prevent further mRNA
degradation.

8. Types of cell-
free extracts
System Advantages Disadvantages
E. Coli •Very high protein yield
•Relatively tolerant of
additives
•Many eukaryotic proteins
insoluble upon expression
•Eukaryotic co- and post-
translational modifications
not possible
•Codon usage is different
from eukaryotes
Rabbit •Mammalian system
•Cap independent
translation
•Sensitive to additives
•Protein glycosylation not
possible
•Co-expression of off-target
proteins
Wheat germ •Translation of large proteins
possible
•Devoid of off-target
endogenous mammalian
proteins
•High protein yield
•Mammalian co- and post-
translational modifications
are not possible
•Premature termination of
products

9. Types of cell-
free extracts
Insect •Translation of large
proteins possible
•No endogenous
mammalian proteins
•Certain forms of
protein glycosylation
possible
•Non-mammalian
Human •Human system
•Co- and post-
translational
modifications are
possible
•Synthesis of functional
proteins
•Possible to make virus-
like particles (VPLs)
•Sensitive to additives
•Lower yields than E.
coli
•New system

10. Cell-Free
Protein
Expression

11. Rabbit
Reticulocyte
Lysate
System
 Rabbit Reticulocyte Lysate (RRL), Nuclease-Treated, is optimized
for mRNA translation by the addition of several supplements.
 In RRL translation reactions, mRNA is used as template for
translation.
 The reticulocytes are purified to remove contaminating cells,
which could otherwise alter the translational properties of the final
extract.
 The lysate contains the cellular components necessary for protein
synthesis (tRNA, ribosomes, amino acids, initiation, elongation
and termination factors).
 Rabbit Reticulocyte Lysate may contain a variety of post-
translational processing activities, including acetylation,
isoprenylation and some phosphorylation activity.

12. Rabbit
Reticulocyte
Lysate
System

13. Application
 Experiments to characterize protein–protein interactions and
protein–nucleic acid interactions
 Rapid and high-throughput expression of mutant or truncated
proteins for functional analysis
 Expression of mammalian proteins with proper glycosylation and
native post-translational modifications (PTMs)
 Labeling of proteins with stable isotopes for structural analysis
 Production of functional virons or toxic polypeptides
 Analysis of components required for protein folding, protein
stability or protein degradation

14. Advantages
 Important tool for molecular biologists in basic and applied
sciences.
 Increasingly being used in high-throughput functional genomics
and proteomics, with significant advantages compared to protein
expression in live cells.
 Essential for the generation of protein arrays, such as nucleic acid
programmable protein array (NAPPA) and enzyme engineering
using display technologies.
 Cell-free approach provides the fastest way to correlate
phenotype (function of expressed protein) to genotype.
 Indispensable for the expression of toxic proteins, membrane
proteins, viral proteins and for proteins that undergo rapid
proteolytic degradation by intracellular proteases.

15. Disadvantages  Cell-free systems are generally not practical for large-scale protein
expression.