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Review
Abstract
Expression and purication of proteins in recombinant DNA systems is a powerful and widely used technique. Frequently there
is the need to express the protein of interest as a fusion protein or chimeric protein. Fusion protein technology is frequently used to
attach a signal which can be used for subsequent localization of the protein or a carrier which can be used to deliver a
therapeutic such as a radioactive molecule to a specic site. In addition to these applications, fusion protein technology can be
employed for several other useful purposes. Of these, the most frequent reason is to provide a tag or handle which will aid in the
purication of the protein. Another useful purpose is to improve the expression or folding of the protein of interest. In these latter
two situations, it is often necessary to remove the fusion partner before the recombinant protein of interest can be used for further
studies. This removal process involves the insertion of a unique amino acid sequence that is susceptible to cleavage by a highly
specic protease. Thrombin and factor Xa are the most frequently used proteases for this application. The purpose of this review is
to discuss the application of thrombin and factor Xa for the cleavage of fusion proteins. It is emphasized that while these enzymes
are quite specic for cleavage at the inserted cleavage site, proteolysis can frequently occur at other site(s) in the protein of interest. It
is necessary to characterize the protein of interest after cleavage from the anity label to assure that there are no changes in the
covalent structure of the protein of interest. Examples are presented which describe the proteolysis of the protein of interest by either
factor Xa or thrombin.
2003 Elsevier Science (USA). All rights reserved.
anity matrix that when used under appropriate conditions will bind and release the tag. Once the chimeric
molecule has been isolated, it is usually necessary to
remove the tag before subsequent use of the expressed
protein of interest [11]. The most popular method to
remove the tag involves the use of a specic protease
such as thrombin or factor Xa. This involves the insertion of a unique amino acid sequence that is specic for
cleavage by either factor Xa or thrombin between the
protein of interest and the fusion partner. In the current
article, we review the use of this technology.
Both thrombin and factor Xa are trypsin-like serine
proteases which will cleave peptide bonds on the carboxyl side (P01 ) of a basic amino acid residue. While
trypsin will eectively cleave peptide bonds when either
lysine or arginine precedes the carboxyl group, thrombin
1046-5928/$ - see front matter 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S1046-5928(03)00168-2
Table 1
Sequences cleaved by thrombin in polypeptide hormones
Polypeptide homonesa
Sequence cleaved
Secretin
Secretin
ELSLSRLRDSA
ELSLSRLR (much
slower than above)
DNYTRLRK
YTRLRKQM
APSGRVSM
VSMIKNLQ
RIRPKLKW
AMAPRERK
NFFWKTFT
KMYPRGNH
QTYPRTNT
a
The reaction mixtures contained 0.5 NIH units thrombin and
1.0 nmol peptide in 20 lL of 50 mM NH4 CO3 , pH 8.0, at 25 C. The
conditions were designed to obtain an enzyme/substrate ratio of 1:60
(w/w).
Table 2
Thrombin-catalyzed cleavage of fusion proteins
Protein of interest
Fusion
protein
ADP-ribosylation
factor
GSTa
b2 -Adreno-receptor
His-tagb -GSa
Transforming
protein E7
GSTa
CRaf1
MEK1
ERK2
Murine osteopontin
His-tagb
GSTa
GSTa
GSTa
HTom20
GSTa
Cystic brosis
transmembrane
conductance
regulator
components
Chlorocatechol
1,2-dioxygenase
Tenascin-C and
Tenascin-R
GSTa
MRP14
IL-6
GSTa
IgG1 FC
QPs3
GSTa
Heavy chain of
tetanus toxin
MBPj
Procathepsin L
GSTa
ORF-14
GSTa
POMC
GSTa
COOH-terminal
domain of
apolipoprotein-E
ACK-42
Deoxyribonucleoside
kinase
His-tagb
Cleavage sequence
SRGSLDPRSFLLRN
PNDKYEPFWEDEEc
His-tagb
GSTa
GSTa
GSTa
GSTa
LVPRGS
LVPRGS
LVPRG
Cleavage condition
Reference
[52]
[53]
[54]
[55]g
[55]g
[55]g
[56]
[57]
[58]
[11]
[59]
[18]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
Table 2 (continued )
Protein of interest
Fusion
protein
Prion domain of
yeast Ure2P
GSTa
Nucleotide-binding
domains of human
multidrug
resistanceassociated protein
(MRP1)
Nucleotide-binding
domains of human
multidrug
resistanceassociated protein
(MRP1)
Human ribosomal
protein S3a
Cleavage sequence
Cleavage condition
Reference
[70]
GSTa
[71]
MBPj
[71]
GSTa
[72]
GST, glutathione-S-transferase.
His-tag is His6 .
c
This is a relatively long cleavage sequence derived from the thrombin platelet receptor sequence and the anion-binding exosite-binding domain
[73]. The thrombin cleavage site in this sequence is at the Arg-8/Ser-9 bond.
d
The fusion protein containing the b2 -adrenoreceptor-GSa protein with or without the thrombin cleavage sequence was obtained as a membrane
preparation.
e
Thrombin was obtained from Haematologic Technologies (Essex Junction, VT).
f
Thrombin was obtained from Sigma Chemical (St. Louis, MO).
g
This study described the development of an interesting assay system for the Raf/MEK/ERK kinase cascade.
h
The thrombin used in this study was from a crude commercial source.
i
The thrombin used in this study was obtained from CalBiochem (San Diego, CA).
j
MBP, maltose-binding protein.
k
Thrombin was obtained from New England Biolabs (Beverly, MA).
l
Thrombin was obtained from AmershamPharmacia Biotech (Piscataway, NJ).
m
The use of room temperature to describe reaction conditions should be avoided. It is assumed that room temperature is 23 C.
n
It was not possible to isolate the nonfused version of the NPD2 protein from the GST fusion protein due to proteolytic degradation.
b
zwitterionic and neutral detergents inhibited the cleavage of fusion proteins byTEV.
Approaches that involve the use of self-cleaving
technology have also been advanced. Hung and Chiou
[37] studied the expression of a serine protease (Tm-5)
from Trimeresurus mucrosquamatus (Taiwan habu) in
E. coli. A cleavage sequence for this serine protease was
inserted between the poly-His coding sequence and the
50 -end of the Tm-5 clone. Renaturation of the protein
after expression demonstrated that the protease had
refolded correctly and cleaved the sequence between the
His tail and the protease.
Hall and Kunkel [38] prepared a triple domain fusion
protein represented by MHL1-VMA1-CBD, where
MHL1 is the MutL homolog protein of interest, VMA1
is a splicing element (or intein), and CBD is the chitin
binding domain from Bacillus circulans. After expression
in a yeast system (Saccharomyces cerevisiae), a soluble
cellular extract was applied to a chitin column which
bound the construct through the chitin binding domain
(CBD). After washing the column to remove unwanted
material, self-cleavage of intein from the MHL-1 protein
was induced by the addition of dithiothreitol.
Table 3
Factor Xa-catalyzed cleavage of fusion proteins
Protein of interest
Fusion partner
ADP-ribosylation
factor
MBPa
MBPa
Archaeoglobus fulgidus
D -lactate
dehydrogenase
Soybean prolin
MBPa
MBPa
D -Glutamate-adding
MBPa
Cleavage
sequence
IEGRISE
IEGR
Cleavage conditions
Reference
[52]
[62]
[74]
[75]
[78]
[76]
[77]
enzymes
Single-chain brinspecic antibodyg
Streptokinase
His-tagi
IQGR
His-tagi
IEGR
p42IP4
Glutathione-Stransferase
Plasminogen
N-terminal peptide
Human glandular
kallikrein
His-tagi
IEGR
HK
Propeptiden
IEGR
Prohevin
E. coli enzyme I
N-terminal domain
Staphylokinase
MBPa
MBPa
IEGR
His-tagi
IEGR
[86]
Nitrogen assimilation
control protein
MBPa
IEGR
Hepatitis B virus e
antigens
Procathepsin L
GlutathioneS-transferase
MBPa
[63]
Tyrosine hydroxylase
type I
Toxoplasma gondii
ROP2 fragment
Plasmodium falciparum
acidicbasic repeat
antigen
Several proteins from
E. colip
MBPa
[91]
[92]
Hirudin
IEGR
MBPa
His-tagi
Ribosomal protein L5
Various fusion
proteins with
his-tagi
MBPa
Na /glucose
cotransporter(SGLTI)
FKBP and Spo0Fq
GlutathioneS-transferase
His-tagi
IEGR
IEGR
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[20]
[88]
[89]
[90]
[93]
[94]
Table 3 (continued )
Protein of interest
Fusion partner
Cleavage
sequence
Cleavage conditions
Reference
29 replication protein
P1
MBPa
IEGR
[95]
His-tagi
IEGR
Hypodermin C
IEGR
C1q
Glutathione-Stransferase
MBPa
Parabutoxin 3
MBPa
IEGR
Human b-endorphin
Outer
membrane
protein F
MBPa
IEGR
IEGR
[96]
[97]
[98]
[99]
[100]
[101]
Comment
The expression of recombinant proteins as fusion
proteins where the fusion partner facilitates purication
is a very useful technology. However, it is clear that
rigorous characterization of the protein of interest following cleavage is essential and this is rarely done. Retention of biological activity does not necessarily mean
that unwanted proteolysis has not occurred during the
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[79]
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11