In vitro translation

Introduction

• In vitro protein expression is the production of

recombinant proteins in solution using biomolecular
translation machinery extracted from cells. Because protein
synthesis occurs in cell lysates rather than within cultured
cells, the method is also called cell-free protein expression.
Introduction (cont)
• In vitro protein expression is a technique that enables
researchers to rapidly express and manufacture small
amounts of functional proteins and also known as
In vitro translation
Cell-free protein expression
Cell-free translation
Cell-free protein synthesis
Applications for which cell-free protein
expression is ideal

• 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)
Applications for which cell-free protein
expression is ideal (cont)

• 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
Applications for which cell-free protein
expression is ideal (cont)
• The diagram provides an example of application of a
cell-free protein expression method combined with
protein mass spectrometry (MS).
Basic components for protein synthesis

• Two basic components are needed to accomplish in

vitro protein expression
1. The genetic template (mRNA or DNA) encoding the
target protein
2. A reaction solution containing the necessary
transcriptional and translational molecular machinery
Cell extract components

• Cell extracts supply all or most of the molecules of the

reaction solution, including:

RNA polymerases for mRNA transcription

Ribosomes for polypeptide translation
tRNA and amino acids
Enzymatic cofactors and an energy source
Cellular components essential for proper protein folding
Cell extract components (cont)
Cell extract components (cont)

• 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”).
Types of cell-free extracts

• Extracts used for cell-free protein expression are made

from systems known to support high level protein
synthesis, some of them described below

E. coli
Wheat germ
Rabbit reticulocytes
Insect cells
Types of cell-free extracts(cont)

• Lysates from E. coli and wheat germ are devoid of

endogenous genetic messages. By contrast, lysates made
from rabbit reticulocytes and insect cells contain
endogenous mRNAs that can be translated during the
synthesis reaction.
• 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.
 Post-translational modifications and human
proteins

• Cell-free systems based on E. coli, RRL and wheat germ

extracts provide some benefits over traditional in
vivo protein expression, they are nonetheless limited in
their ability to produce human proteins complete with post-
translational modifications (PTMs).
• Cell-free systems from E. coli and wheat germ extracts are
not capable of protein glycosylation.
Post-translational modifications and human
proteins

• Rabbit reticulocyte systems, canine microsomal

membranes must be added to the translation mix to
produce glycosylated proteins but decrease overall
protein yield.
• Glycosylated protein yields from insect cell-free systems
are higher than from RRL systems, but the glycosylation
patterns obtained are different from those produced by
human cells.
 Transcription and translation in cell-free
expression

• Cell-free expression systems can support protein

synthesis from DNA templates (transcription and
translation) or mRNA templates (translation only).
• In principle, cell-free expression systems can be
designed to accomplish transcription and translation
steps as two separate sequential reactions (linked) or
concurrently as one reaction (coupled).
 Role of in vitro translation in functional
genomics and proteomics in Future

In the post-genomic era, cell-free protein synthesis has the

potential to become one of the most important high
throughput technologies for functional genomics and
proteomics.

• It is the quickest way to obtain an expressed phenotype

(protein) from a genotype (gene).

• It is independent of host cells. Proteins which are toxic or

prone to proteolytic degradation can be readily prepared in
vitro
 Role of in vitro translation in functional
genomics and proteomics in Future(cont)

• Commercially available cell-free protein synthesis systems

are typically derived from cell extracts of Escherichia coli
S30, rabbit reticulocytes or wheat germ.
References

• Imataka H, Mikami S (2009) Advantages of human cell-

derived cell-free protein synthesis
systems. Seikagaku 81(4):303–7.
• Mikami S et al. (2008) A human cell-derived in
vitro coupled transcription/translation system optimized
for production of recombinant proteins. Protein Expr
Purif 62(2):190–8.
• Kobayashi T et al. (2007) An improved cell-free system for
picornavirus synthesis. J Virol Methods 142(1-2):182–8.
References (cont)

• Mikami S et al. (2006) A hybridoma-based in

vitro translation system that efficiently synthesizes
glycoproteins. J Biotechnol 127(1):65–78.
• Mikami S et al. (2006) An efficient mammalian cell-
free translation system supplemented with translation
factors. Protein Expr Purif 46(2):348–57.
• Kaiser, C.M., et al. (2006) Real-time observation of
trigger factor function on translating ribosomes.
Nature, 444, 455–460.
• Graslund, S., et al. (2008) Protein production and
purification. Nat. Methods, 5, 135–146.