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Previous Article | Next Article 
J Virol, April 1998, p. 3383-3386, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Efficient Expression and Rapid Purification of
Human T-Cell Leukemia Virus Type 1 Protease
Y. Shirley
Ding,1
Sherry M.
Owen,1,2
Renu B.
Lal,2 and
Richard A.
Ikeda1,*
School of Chemistry and Biochemistry, Georgia
Institute of Technology, Atlanta, Georgia
30332-0400,1 and
Retrovirus Diseases
Branch, Division of Viral and Rickettsial Diseases, Centers for
Disease Control and Prevention, Atlanta, Georgia
303332
Received 24 September 1997/Accepted 12 December 1997
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ABSTRACT |
Human T-cell leukemia virus type 1 (HTLV-1) is an oncovirus that is
clinically associated with adult T-cell leukemia. We report here the
construction of a pET19-based expression clone containing HTLV-1
protease fused to a decahistidine-containing leader peptide. The
recombinant protein is efficiently expressed in Escherichia coli, and the fusion protein can be easily purified by affinity chromatography. Active mature protease in yields in excess of 3 mg/liter of culture can then be obtained by a novel two-step refolding
and autoprocessing procedure. The purified enzyme exhibited Km and Kcat values of
0.3 mM and 0.143 sec
1 at pH 5.3 and was inhibited by
pepstatin A.
| |
TEXT |
Human T-cell leukemia virus type 1 (HTLV-1) is an oncovirus in the Retroviridae family
(11). HTLV-1 was first isolated in the early 1980s from
patients with adult T-cell leukemia/lymphoma (13) and has
been subsequently shown to be clinically associated with adult T-cell
leukemia/lymphoma (16), HTLV-1-associated myelopathy/tropical spastic paraparesis (3), and a number of other chronic diseases (e.g., uveitis, arthritis, and infective dermatitis) (2, 8).
Although the HTLV-1 protease gene has been cloned and the enzyme has
been expressed and purified, the activity of the protease is not
well characterized. The problems that have been previously encountered in the characterization of HTLV-1 protease can probably be
attributed to a lack of sufficient quantities of purified enzyme. The
highest yields of purified, recombinant HTLV-1 protease that have been
reported to date are 350 µg per liter of culture (9). We
report here the expression and purification of recombinant HTLV-1
protease at yields of 3.0 mg/liter, the characterization of the
catalytic activity of HTLV-1 protease, and the inhibition of HTLV-1
protease by pepstatin A.
Construction of a plasmid that expresses HTLV-1 protease.
A
DNA fragment containing the reading frame for the HTLV-1 protease
precursor (base pairs 2073 to 2778 of the HTLV-1 MT-2 sequence) was
obtained by amplification of HTLV-1 DNA (10, 14) with
Taq DNA polymerase and primers 1 and 2 (Table
1). The purified fragment was digested
with EcoRI and XhoI and ligated into the corresponding sites of pGEX4T-1 (Pharmacia) to obtain the plasmid pPR100. In order to simplify the purification of HTLV-1 protease, the
putative protease coding sequence was cloned into a plasmid, pET19b,
that encodes an expression leader containing 10 consecutive histidines
(12). A DNA fragment containing mature HTLV-1 protease (9) was obtained by amplifying pPR100 with Taq
DNA polymerase and primers 3 and 4 (Table 1). These primers were
designed to add an in-frame BamHI site to the 5' end of
HTLV-1 protease (nucleotide CCAGTT), to introduce a stop
codon at nucleotide 2531 (between ATCTTG and CCAATA),
and to add a BamHI site to the 3' end of the gene. The
locations of the 5' BamHI site and the stop codon with respect to the HTLV-1 coding sequences were based on the previously reported identities of the N and C termini of HTLV-1 protease (9). The PCR fragment was then inserted into the
BamHI site of pET19b to construct the expression plasmid
pPR101 (Fig. 1).
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FIG. 1.
The expression vector and predicted amino acid sequence
of HTLV-1 protease. (A) Plasmid pPR101. Ori, origin of replication;
Amp, ampicillin resistance gene; T7/lac, T7 promotor; LacI,
lacIq gene; His-prt, HTLV-1 protease sequences
fused to the histidine tag of pET19b. (B) The predicted amino acid
sequence of the recombinant HTLV-1 protease fusion protein. The
autoprocessed site is between amino acids 27 and 28 (between D and
P).
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Expression and purification of an HTLV-1 protease fusion
protein.
Cultures (30 ml) of pPR101/Escherichia coli
BL21(DE3)pLysS were grown at 37°C in LB/Amp to an optical density
at 600 nm of 0.6. The culture was then induced by the addition of
isopropyl-
-D-thiogalactopyranoside (IPTG) (0.4 mM final
concentration). Three hours after the addition of IPTG, the cells were
harvested by centrifugation, resuspended in buffer A (20 mM Tris, pH
7.9, 5 mM imidazole, 500 mM NaCl), and sonicated. The bacterial lysate
was cleared by centrifugation, and the pellet was resuspended in buffer
B (buffer A plus 8 M urea). The mixture was cleared by centrifugation,
and the supernatant was then loaded on a 1 ml His-Bind column
(Novagen). The column was then washed with buffer B and buffer C (20 mM
Tris, pH 7.9, 20 mM imidazole, 500 mM NaCl, 8 M urea) and eluted with
buffer D (20 mM Tris, pH 7.9, 1 M imidazole, 500 mM NaCl, 8 M urea)
under denaturing conditions.
Samples from different steps of the purification (Fig.
2) were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and are shown in
Fig. 3. A major band is visible at 20 kDa
in a sample of the lysate pellet. This band corresponds to the expected
molecular size of HTLV-1 protease fused to the 27-amino-acid pET19b
histidine tag (Fig. 1B) and is not observed in samples of E. coli BL21(DE3)pLysS that harbor the parent plasmid pET19b (data
not shown). The 20-kDa protein also bound to the His-Bind resin
affinity column and eluted with imidazole (Fig. 3A).
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FIG. 2.
Flow chart of the purification of HTLV-1 protease from
E. coli. Supernatant I was obtained from centrifugation of
the cell lysate of E. coli BL21(DE3)pLysS/pPR101;
supernatant II was obtained from centrifugation of pellet I redissolved
in buffer B; load I was the fraction collected while His-Bind column I
was being loaded; wash Ia was the fraction collected while washing
His-Bind column I with buffer B; wash Ib was the fraction collected
while washing His-Bind column I with buffer C; elute I was the fraction
collected while eluting His-Bind column I with buffer D; elute I
contains the protease fused to the His-tag leader; processed refolded
protein was obtained after the urea was removed by a two-step dialysis;
redenatured protein was obtained by dissolving the processed protease
fraction in buffer B; load II was the fraction collected while His-Bind
column II was being loaded with redenatured protein; wash IIa was the
fraction collected while washing His-Bind column II with buffer B; wash
IIb was the fraction collected while washing His-Bind column II with
buffer C; elute II was the fraction collected while eluting His-Bind
column II with buffer D; load II and wash IIa contained the mature
protease.
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FIG. 3.
SDS-PAGE analysis of different purification steps. (A)
SDS-PAGE analysis of samples from His-Bind column I. Lanes: 1, low-range molecular size marker (Bio-Rad); 2, 10 µl of supernatant I;
3, 10 µl of supernatant II; 4, 10 µl of load I; 5, 10 µl of wash
Ia; 6, 10 µl of wash Ib; 7, 10 µl of elute I; 8, 10 µl of
processed and refolded protein. (B) SDS-PAGE analysis of samples from
His-Bind column II. Lanes: 1, low-range molecular size marker
(Bio-Rad); 2, 10 µl of redenatured protein; 3, 10 µl of load II; 4, 10 µl of wash IIa; 5, 10 µl of wash IIb; 6, 10 µl of elute II. a,
protease fusion protein; b, processed protease. Molecular size markers
(in kilodaltons) are on the left.
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Autoprocessing produces HTLV-1 protease.
To obtain active
HTLV-1 protease, a novel renaturation and autoprocessing protocol was
developed. The purified fusion protein was refolded by sequential
dialysis against buffer E (10 mM sodium acetate buffer, pH 3.5, and 1 mM dithiothreitol [DTT]) and buffer F (100 mM sodium citrate buffer,
pH 5.3, 5 mM EDTA, 1 mM DTT, and 1 M NaCl). Autoprocessing is observed
during dialysis against buffer F and yields a 14-kDa protease. The
processed protease was then separated from the fusion protein on a
second His-Bind column under denaturing conditions (samples from this
second His-Bind column are shown in the SDS gel in Fig. 3B). The mature
protease ran through the column and was collected, while the
unprocessed fusion protein was retained on the column (Fig. 3B, lanes
3, 4, and 6). Typically, 100 µg of HTLV-1 protease could be purified from a 30-ml culture. (A summary of the purification procedure is shown
in Table 2.)
To confirm the identity of the processed protease, the N terminus of
the purified protein was sequenced. It was observed that the first 12 amino acids of the mature protein were
Pro-Val-Ile-Pro-Leu-Asp-Pro-Ala-Arg-Arg-Pro-Val. This sequence matches
the expected N-terminal sequence of the mature protease and confirms
that the fusion protein was cleaved between the N-terminal leader and
putative mature protease.
Enzymatic properties.
Incubation of the refolded HTLV-1
protease with the synthetic peptide APQVLPVMHPHG (4, 6, 9),
a peptide containing the native cleavage site between P19 and P24 of
the HTLV-1 Gag protein, yielded two new peptide peaks that comigrated
with the chemically synthesized, predicted products APQVL and PVMHPHG. This suggested that the purified HTLV-1 protease was active. The salt
and pH optimums for cleavage of a synthetic peptide by HTLV-1 protease
were found to be 1.0 M and pH 5.3 (data not shown), respectively, as
previously reported (8a, 10a), and further studies showed that the peptide hydrolytic activity of the purified HTLV-1 protease obeyed simple Michaelis-Menten kinetics. The purified enzyme exhibited Km and Kcat values of 0.3 mM and 0.143 sec1 at pH 5.3.
Since HTLV-1 protease is a member of the family of aspartic proteases
(15), inhibition by pepstatin A, a typical inhibitor of
aspartic proteases, was measured. The data showed that pepstatin A can
inhibit the proteolysis of the synthetic peptide substrate, but yields
a Ki value of 200 µM (Fig.
4). This result is significantly higher
than the value obtained by Kobayashi et al. (9), but it is
similar to the data obtained by Daenke et al. (1).
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FIG. 4.
Inhibition of HTLV-1 protease by pepstatin A. A Dixon
plot of the inhibition of HTLV-1 protease by pepstatin A is shown.
Inhibitor concentrations ranged from 0 to 800 µM; substrate
concentrations ranged from 60 to 90 µM.  , 60 µM;  , 75 µM;
 , 90 µM.
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In work by other investigators, HTLV-1 protease has been cloned and
expressed in E. coli either as the precursor protease or the
mature protease (1, 9), but in both cases, expression of
HTLV-1 protease was extremely poor (20 to 350 µg per liter). The
pPR101 expression clone was designed to produce HTLV-1 protease fused
to a leader peptide containing a decahistidine sequence. This was done
to allow for rapid purification of the recombinant protein over a
nickel column; however, purification of the protein was complicated by
the limited solubility of the protease. The recombinant protease fusion
protein precipitates as it is expressed in E. coli and can
only be resolubilized under denaturing conditions. Since the protease
precursor is inactive in an insoluble form, this may reduce the
previously observed toxicity of the protease and improve the yields of
the recombinant protein (5, 7). Fortunately, the denatured
precursor protease is easily purified by affinity chromatography, and
the precursor refolds and autoprocesses itself when the denaturant is
removed by dialysis.
The N-terminal amino acid sequence of the processed protease shows that
precursor protease cleaves itself at the junction of the histidine
peptide leader and the first amino acid of the mature HTLV-1 protease.
The amino acid sequence at this cleavage site is ED/PV which is very
different from the protease cleavage sites seen in the HTLV-1 Gag
protein (VL/PV) and might explain why autoprocessing is not 100%
efficient (Fig. 3A).
In the future we plan to test whether HTLV-1 can be inhibited by a wide
series of human immunodeficiency virus type 1 protease inhibitors and
whether specific inhibitors of HTLV-1 protease can be identified.
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ACKNOWLEDGMENTS |
We thank Nadia Boguslavsky for technical assistance.
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FOOTNOTES |
*
Corresponding author. Mailing address: School of
Chemistry and Biochemistry, Georgia Institute of Technology, 706 State
St., Atlanta, GA 30332-0400. Phone: (404) 894-4037. Fax: (404)
894-7452. E-mail: RICK.IKEDA{at}CHEMISTRY.GATECH.EDU.
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J Virol, April 1998, p. 3383-3386, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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