Efficient Expression and Rapid Purification of Human T-Cell Leukemia Virus Type 1 Protease

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 K_m and K_cat values of 0.3 mM and 0.143 sec⁻¹ at pH 5.3 and was inhibited by pepstatin A.

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).

TABLE 1.

PCR primers

1a	5′ TCG AAT TCC ATC CCA CAC CCA AA 3′
2a	5′ TGC ACT CGA GTT AGA GAG TTA GTG GCC 3′
3b	5′ AGG GCG GGA TCC AGT TAT ACC GTT AGA T 3′
4b	5′ ATA TAT GGA TCC TCA CAA GAT TAC AGG CGG CCC 3′

^aPrimers 1 and 2 were used to amplify the HTLV-1 protease precursor. The bold text shows the sequence annealing to the HTLV-1 sequences. Lightface text indicates the sequence tail containing the added restriction sites. 

^bPrimers 3 and 4 were used to amplify the mature HTLV-1 protease. The bold text shows the sequence annealing to the HTLV-1 sequences. Lightface text indicates the sequence tail containing the added restriction sites. Italics indicate the stop codon introduced at the C terminus of the mature protease. 

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, lacI^q 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).

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-β-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).

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.

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.

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.)

TABLE 2.

Purification scheme

Step	Volume (ml)	Total protein (mg)a	Total activity (mU)b	Sp act (mU/mg)	Yield (%)
Extract	6	11	—c	—	—
His-Bind column I	6	1.1	34.76	31.6	100
His-Bind column II	7	0.103	22.2	216	64

^aProtein concentration was determined by Bradford protein assays (Bio-Rad). Bradford assays with bovine serum albumin as the standard are known to underestimate the concentration of pepsin. Therefore, pepsin was used as the standard for determining the concentration of HTLV-1 protease, since pepsin belongs to the same aspartyl protease family as HTLV-1 protease. 

^bOne unit of enzyme activity was defined as the amount of enzyme that cleaves 1 mmol of synthetic peptide/min in the standard assay (9). 

^c—, unmeasurable due to the presence of urea. 

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 K_m and K_cat values of 0.3 mM and 0.143 sec⁻¹ 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 K_i 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).

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.

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.