- Email: [email protected]
Assay of HIV-1 protease activity by use of crude preparations of enzyme and biotinylated substrate
Journal of Virological Methods ELSEVIER
Journal of Virological
Methods 53 (1995) 63-73
Assay of HIV-l protease activity by use of crude preparations of enzyme and biotinylated substrate Sung-Liang Yu, Nay Wang, Chiou-Yi Liou, Wan-Jr Syu* Graduate Instifute of Microbiology and Immunology, National Yang-Ming Uniuersity, Shih-Pai, 112, Taipei, Taiwan, ROC Accepted 6 December
1994
Abstract An enzyme immunoassay was developed for monitoring protease reactions of human immunodeficiency virus (HIV). The protease and its substrate, the gag precursor, were generated separately in Escherichia coli. The HIV-l protease was generated with a glutathione-S-transferase expression system and the gag substrate, named Pin17/24, was prepared with a Pinpoint expression system. Pin17/24 consists of an N-terminal peptide, which is biotinylated in E. coli, fused with a C-terminal peptide that contains a protease cleavage site flanked by p17 and p24 segments. Through its biotin in the N-terminal region, Pin17/24 bound to ELISA plates coated with avidin, whereas through its C-terminal region, the same molecule of Pin17/24 could be recognized b’y an anti-p24 monoclonal antibody. When the protease was added to Pin17/24, the p24 fragment was released from the biotinylated fusion protein and could no longer be retained on the avidin plates, and as a result, binding of the anti-p24 monoclonal antibody decreased. The binding was specific and the reaction was inhibited by a known HIV protease inhibitor. Due to the specific interactions between avidin and biotin, monoclonal antibody and antigen, and the HIV protease and1the gag substrate, crude preparations of these reagents can be used readily in the assay. The simplicity and feasibility of this method should be useful for simultaneous monitoring of many enzyme reactions, particularly for screening possible HIV protease inhibitors. Keywords: Human immunodeficiency ferase; Biotin
l
Corresponding
0166-0934/95/$09.50
virus; Protease; ELISA, Inhibitor;
author. Fax 886 2 821 2880. 0 1995 Elsevier Science B.V. All rights reserved
SSDI 0166-0934(94)00177-4
Fusion protein; Glutathione-S-trans-
64
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
1. Introduction Human immunodeficiency virus (HIV) is the primary agent for the acquired immunodeficiency syndrome (Levy, 1993). Like other retroviruses, HIV encodes an aspartic protease that is responsible for the processing of the viral gag and gag-pol polyproteins. A functionally active protease is essential for the replication and infectivity of HIV. The HIV protease therefore has been recognized as one of the viral target enzymes for anti-HIV therapy (Johnston and Hoth, 1993; Wlodawer and Erickson, 1993). Several inhibitors against this enzyme have been developed and possible usage in patients is being evaluated (Johnston and Hoth, 1993; Lam et al., 1994). Since HIV has high mutation rates and since drug resistant mutants emerge within months after receiving reverse transcriptase inhibitors (Larder et al., 19931, the virus may also evolve quickly into mutants that are resistant to protease inhibitors (Johnston and Hoth, 1993; Otto et al., 1993). One way of dealing with the problem of drug resistance is to develop continuously new drugs. An economic and convenient method for measuring the viral enzyme activity should accelerate the discovery of novel anti-HIV reagents. Most methods used to monitor the protease activity can be divided into two categories. One is to use synthetic peptides or peptide analogs that mimic the junction regions in the native substrates, gag and gag-pol polyproteins (Billich et al., 1988; Darke et al., 1989; Boutelje, et al., 1990; Cheng et al., 1990; Matayoshi et al., 1990; Wondrak et al., 1990; Broadhurst et al., 1991; Lam et al., 1994). The peptide cleavage is detected by high performance chromatography or monitored by absorbance changes. In these systems, the proteases used are purified. Furthermore, these approaches may have to be substantiated with natural substrate reactions (Billich et al., 1988; Kotler et al., 1988). The second category of methods is to use the natural gag substrates expressed in E. coli. The substrates and the enzymes used could be obtained directly from crude bacterial lysates. The enzymatic reactions are monitored by Western blotting analysis of the proteolytic products generated and separated from the substrate precursor (Giam and Boros, 1988; Overton et al., 1989; Dilanni et al., 1990; Grant et al., 1991; Kotler et al., 1992). While the specificity of the reaction can be visualized directly on blots, immunoblotting is tedious and the setting is difficult for simultaneous measurements of numerous reactions. A simple and feasible enzyme-linked immunosorbent assay (ELISA) for detecting HIV-l protease activity is described. A purification step is not necessary for the preparations of the protease, the recombinant substrate, and the monoclonal antibody (MAb) used.
2. Materials and methods 2.1. DNA and immunochemical
reagents
All plasmid transformations were carried out in E. coli strain JM109. DNA manipulations were according to the published protocols (Sambrook et al., 1989). Plasmids after ligation and transformation were checked by restriction enzyme mapping and confirmed further by DNA sequencing of the fusion junctions.
S.-L. Yu et al. /Journal of Virological Methods 53 (1995) 63-73
65
Avidin, avidin-horseradish peroxidase (HRP) conjugates, alkaline phosphatase-labeled goat anti-mouse IgG (whole molecule), and HRP-labeled goat anti-mouse IgG (whole molecule) were obtained from Sigma (St Louis, MO). MAb-containing ascites fluids from BALB,/c mice were used directly without further purification. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Hybridoma 183 (Clone H12-5C), producing MAb against HIV-l ~24, from Dr. Bruce Chesebro. 2.2. Generations of the HlV-1 protease and Pin1 7/24
fusion protein
To produce HIV-l protease, a BgZII/ScaI-restricted fragment of proviral HIV-l DNA HXB2 (nucleotides 2096-2753) (Ratner et al., 1985) was ligated to BamHI/SmaI-digested pGEX-1 (Pharmacia, Uppsala, Sweden). The resulting plasmid pGEXpr encodes a pol-encoded fragment fused to the carboxyl terminus of the glutathione S-transferase (GST) from Schistosoma japonicum (Smith and Johnson, 1988). When induced with isopropyl+?-D-thiogalactopyranoside (IPTG), active HIV-l protease was produced by autocatalytic releasing (Giam and Boros, 1988) from the GST fusion protein (Fig. 1A). To express a gag analog as the HIV-l protease substrate, a Hind111 fragment of HXB2 (nucleotides 1085-1712) was cloned into pBluescript II SK+ (Strategene, La Jolla, CA). The resulting plasmid with the right orientation of insert was then digested with BamHI and HincII. A 0.7-kb fragment was isolated and ligated with the BamHI/EcolRV-restricted Pinpoint Xa-1 vector (Promega, Madison, WI). The expression vector so obtained encoded an about 40 kDa recombinant protein, named Pin17/24. Pin17/24 contains an N-terminal peptide, which is biotinylated at a single lysine residue in E. coli (Promega Technical Manual), fused with a C-terminal gag fragment that consists of a protease cleavage site flanked by p17 and p24 segments (Fig. 1B) 2.3. Preparations of protease and substrate Plasmid-transformed E. coli were cultured in LB broth in the presence of ampicillin (50 pg/ml:l and induced with 1 mM IPTG at 37°C for l-3 h when the optical density at 570 nm reached 0.5. Typically, bacteria of 300 ml culture were pelleted and resuspended in 6 ml substrate buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM P-mercaptoethanol) or 6 ml protease buffer (50 mM Na-acetate pH 5.0, 0.35 M NaCl, 5 mM EDTA, 1 mM PMSF, and 10 mM P-rnercaptoethanol). Bacteria were then lysed with French pressure cell press (SLM Instruments, Urbana, IL). After centrifugation at 6000g for 30 min at 4°C to remove cell debris, the supematants were used directly as the sources for the HIV-l protease or the protease substrate. 2.4. Enzymatic reactions Western blotting was carried out using 4 ~1 of the Pin17/24 lysate mixed with 2 ~1 of the protease lysate and incubated for 1 h at 37°C. The reaction was stopped by adding
66
S. -L. Yu et al. /Journal of Virological Methods 53 (I 995) 63-73
Protease (B)
Biotin
7
I_(
*
~24 fragment
Pin1 7124 Protease M.W.66 45 36 29 24
-
20 -
14 Fig. 1. HIV-l protease generation and reactions with recombinant substrate Pin17/24. (A) Diagram for the HIV-1 protease autocatalytically released from the GST fusion protein (see Section 2, Materials and methods). (B) Illustration for the Pin17/24 recombinant protein and the products after HIV-1 protease digestion. The arrow heads indicate the HIV-1 protease cleavage sites; thin lines represent the sequences derived from the fusion vector sequences whereas thick lines represent that derived from HIV-1 coding sequences. The relative location of biotin on Pin17/24 is also labeled. (C) Western blotting analysis of Pin17/24 with and without HIV-l protease treatment; detection agents used were avidin-HRP and anti-p24 MAb, respectively. F labels the full length Pin17/24. After the HIV-1 protease digestion, Pin17/24 should yield two fragments as illustrated in (B). N labels the 17 kDa N-terminal product with the biotin tag that was detected only by avidin-HRP; C labels the 23 kDa C-terminal fragment that was detected only by the anti-p24 MAb. See Section 4 (Discussion) for explanation of additional bands.
6 pl of 2 X sodium dodecyl sulfate (SDS) sample buffer and boiled. Proteins in the mixtures were separated by SDS-containing polyacrylarnide gel electrophoresis, electroblotted to nitrocellulose filters. The blots were blocked with 3% gelatin in TBS (0.1 M Tris, pH 7.5, 0.5 M NaCl) for 1 h, reacted with avid&conjugated horseradish peroxidase (HRP), and developed with 4-chloro-1-naphthol and H,O, as previously
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
67
described (‘Yu et al., 1993). Alternatively, blots were reacted sequentially with the anti-p24 MAb and HRP-labeled goat anti-mouse antibodies. Blots were then developed accordingly. For ELISA, plates were first coated with 100 ~1 avidin (10 pg/ml in 0.1 M Tris, pH 9.0, 0.15 M NaCl) overnight. After washing with TBS, plates were blocked with 3% bovine serum albumin (BSA) in TBS for 1 h. Plates were used immediately or washed three times with TBS, air-dried, and stored at 4°C until use. Protease reactions in triplicates were performed by mixing 4 ~1 of the Pin17/24 lysate with 2 ~1 of appropriately diluted protease lysates for 1 h at 37°C. 94 ~1 of anti-p24 MAb solutions (ascites fluid 1 to 500 diluted in TBS containing 1% BSA) were added to the mixtures. After a brief mixing, the mixtures were transferred immediately to wells of avidin-coated plates. After incubation at room temperature for 1 h, plates were washed 5 times with TBS. Alkaline phosphatase-conjugated goat anti-mouse antibodies (1 pg/ml in 1% BSA-TBS) were added (100 pi/well) and incubated for an additional 1 h. Plates were then washed 5 times with TBS and developed with 4-methylumbelliferyl phosphate (Sigma) (Syu and Kahan, 1991). The relative fluorescence intensity after alkaline phosphatase hydrolysis was measured using CytoFluor 2300 (Millipore Asia, Taiwan). Pepstatin A (Sigma) was dissolved in dimethyl sulfoxide (DMSO) as a 7 mM stock. Appropriat’e concentrations were made by diluting the stock with DMSO. For a control, an equivalent amount of DMSO was added to the protease reaction.
3. Results To demonstrate that Pin17/24 is an appropriate substrate for HIV-l protease, Western blotting analysis was used with both avidin-HRP and the anti-p24 MAb to monitor the proteolytic reactions (Dilarmi et al., 1990). Consistent with the prediction from the fusion protein construct, Pin17/24 (labeled F in Fig. 1C) has an apparent molecular weight of about 40 kDa and was recognized by both avidin-HRP and the anti-p24 MAb. When HIV-l protease was added to the reaction, Pin17/24 was digested and no longer detected by avidin-HRP. However, the anti-p24 MAb still detected the residual Pin17/24 molecules, indicating that a complete digestion had not been reached and that the anti-p24 MAb detection had a slightly higher sensitivity than that of avidin-HRP. After the HIV-l protease reaction, two digestion products should be generated from Pin17/24 as illustrated in Fig. 1B; the biotinylated N-terminal fragment should be detected only by avidin-HRP while the C-terminal fragment is to be detected only by the anti-p24 MAb. As shown in Fig. 1C a newly generated 17 kDa product (labeled N) was detected only by avidin-HRP and another 23 kDa product (labeled C) was detected only by the anti-p24 MAb. Therefore, these two newly generated products may represent the N-terminal fragment and the C-terminal fragment of the protease-digested Pin17/24, respectively. To adapt Western blotting analysis into a convenient ELISA format, the aim was to simplify the reagent preparations. It was reasoned that plate-coated avidin will bind biotinylated recombinant protein expressed in a crude bacterial lysate with high affinity so that any attempt to purify Pin17/24 is unnecessary. Second, since Pin17/24 has a
68
S.-L. Yu et al. /Journal
1 If; >
of Virological Methods 53 (1995) 63-73
Biotinylated substrate (Pin17/24)
Anti-p24 MAb
Protease
Alkaline phosphatase-conjugated goat anti-mouse Ab
Protease inhibitor (pepstatin A)
R r
(A) Buffer only (B) Protease (C) Protease + Inhibitor -
R4
Fig. 2. A schematic drawing of ELISA measurements of protease reactions. HIV-l protease digestron of Pin17/24 was carried out in microtest tubes for 1 h at 37°C. Solutions with the anti-p24 MAb were then added and the whole mixtures were immediately transferred to avidin-coated ELISA plates. Plate-coated avidin bound the biotin tag in the N-terminal region of Pin17/24, which was in turn recognized by the anti-p24 MAb through the C-terminal region; the plate-retained anti-p24 MAb was then enzymatically measured (see Section 2, Materials and methods) by binding of alkaline phosphatase-conjugated goat anti-mouse IgG (A). When the HIV-1 protease cleaves Pin17/24 into the N-terminal biotin-tagged fragment and the C-terminal p24 fragment, the anti-p24 MAb bound to the p24 fragment could no longer be retained on the plates during the next washing (B). In the presence of an HIV-l protease inhibitor, binding of the anti-p24 MAb to the plates is retained CC).
single biotin in the N-terminal region and a p24 segment in the C-terminal region, simultaneous binding of avidin and the anti-p24 MAb may be possible. Third, a high signal/ noise ratio is highly preferable in the assays. Prior binding of Pin17/24 to plates could restrict the molecular freedom and decrease the accessibility of P17/24 to the HIV-l protease. As a result, the signal/noise ratio of the detection may decrease. To avoid this possibility, the solutions were mixed and the digestion was carried out in microtest tubes. After 1 h digestion, the anti-p24 MAb was added to the tubes and the whole mixtures were transferred to avidin-coated plates. Thereafter, reactions were carried out in the ELISA plates. As illustrated in Fig. 2, when HIV-l protease is functioning, the relative ELBA readings should decrease drastically. When HIV-l protease is inhibited, the ELISA readings are expected to be comparable to that of the buffer controls. Typical results are shown in Fig. 3. As reflected by fluorescence intensity generated from the reaction of alkaline phosphatase bound in the immunocomplexes, the anti-p24 MAb bound on plates decreased progressively as more protease lysates were added. The anti-p24 MAb reached base-line binding after sufficient protease was added to cleave all
S.-L. Yu et al. /Journal
+
7
L -0
c
of Virological Methods 53 (1995) 63-73
loo
GST lysate Pmtease
z m -
69
lysate 0
\ h
_
“i 0 \
‘\ i %.. ‘k.‘.*_......0 ..............”.......* .....“.”..........“.O
0
1
4 Bacteial
Lysate *tied
(~1)
Fig. 3. Dose-dependent cleavage of Pin17/24. Increasing amounts of bacterial lysates containing the HIV-l protease or the control GST molecule were incubated with the Pin17/24 bacterial lysate. Proteolytic cleavage was determined by the decrease of the plate-bound anti-p24 h4Ab (see Section 2, Materials and Methods). Each data point represents a mean of triplicate; standard deviations of the readings were about 10%. Repeated experiments gave similar results.
the digestible Pin17/24. In contrast, the control GST lysate, which derived from the pGEX-1 vector-transformed bacteria, did not decrease efficiently the binding of the anti-p24 even at high concentrations. To substantiate further that the ELISA results did reflect the specific cleavage of HIV-l protease, reactions in the presence of a known inhibitor, pepstatin A (Katoh et al., 1987; Seehneier et al., 1988; Richards et al., 1989), were carried out. Western blotting analysis and ELISA were carried out for a direct comparison. When concentrations of pepstatin A were increased, the cleavage of Pin17/24 and the production of 23 kDa product were suppressed increasingly, as shown clearly in the Western blot (Fig. 4A). Consistent with the Western blotting results, ELISA data (Fig. 4B) indicated that the degrees of inhibition increased accordingly. Pepstatin A at a concentration of 200 PM gave 95% .inhibition, which was comparable to the results of others (Katoh et al., 1987; Seelmeier et al., 1988; Richards et al., 1989).
4. Discussiion A rapid and easy assay was developed to monitor the HIV protease activity. This system has a unique property of using crude preparations of the HIV protease, the recombinant substrate, and the anti-p24 MAb. No purification such as ammonium sulfate precipitation, urea extraction, or refolding is necessary. While Western blotting results are good for qualitative assessment, it is tedious to assay quantitatively the degree of inhibition. Moreover, it is difficult to use Western blotting for analyzing dozens of simultanealus reactions. The ELISA method provided results comparable to that of Western blotting analysis as shown in Fig. 4. Furthermore, ELISA allows quantitative measurements of hundreds of simultaneous protease reactions.
70
S.-L. Yu et al. /Journal
of Virological Methods 53 (I 995) 63- 73
Pin17124 + Protease Pepstatin A
0
0
+ +
+ +
+ +
0
10
50
lb
50
+ + 200 WV
200
Pepstatin A (PM) Fig. 4. Inhibition of HIV-l protease activity analyzed by (A) Western blotting and (B) ELISA. Increasing concentrations of pepstatin A were used to inhibit the HIV-1 protease digestion of Pin17/24. Quaternate digestions were conducted; one sample was analyzed with Western blotting and triplicate were used for ELISA analysis. Western blotting was analyzed with the anti-p24 h4Ab as in Fig. 1 while ELISA was done as in Fig. 3. Standard deviations of the ELISA measurements were about 10%. See legend to Fig. 1 for the explanation of labels F and C.
When recombinant proteins are overproduced in E. coli, nonspecific protease degradation frequently occurs. This was also observed in the preparation of Pin17/24. In the blots with crude bacterial lysates, avidin-HRP detected a 20.5 kDa peptide and the anti-p24 MAb detected a 25 kDa peptide in addition to Pin17/24 (Fig. 1C). Also shown in Fig. lC, both peptides were digested by the HIV-l protease, indicating that both fragments were degraded products of Pin17/24 and all retained the HIV-l protease cleavage site. These degradation products and another minor 18 kDa biotinylated
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
71
product, which was not affected by HIV-l protease (Fig. lC), did not interfere with the ELISA measurement. When the cell lysates of HIV-l-infected CEM cells were used as the source of the protease, the enzyme activity could also be monitored. However, this system was sensitive to SDS and deoxycholate (data not shown). As a result, the HIV-infected cells are normally lysed with the protease buffer containing additional 1% Triton X-100. Recently, Mansfeld et al. (1993) described an ELISA using a recombinant protein as the protease substrate. Their method relies on extracting the recombinant proteins from inclusion bodies and refolding it into digestible substrates. The extraction and refolding are time-consuming and difficult to control for the degree of correct folding. In contrast, our Pin17/24 was soluble in the bacterial lysate and protease digestible without any treatment. Pin17/24 was expressed equally well in E. coli either in the presence of IPTG (Fig. 1C) or without this inducer (data not shown). The ‘leakage’ expression of Pin17/24 offers an additional cost-effective advantage to this substrate preparation. An additional difference between this method and that of Mansfeld et al. (1993) is that we have proteolytic reaction occurred in solutions whereas the latter coat the refolded substrate directly onto plates for protease digestion. Since the steric hindrance of the plate cannot be completely excluded, a portion of the substrates coated on plates may not be accessible to the protease. These inaccessible substrates and molecules with incorrect folding may contribute to a high background binding of the Mab and decrease the signal/noise ratio. Indeed their best signal/noise ratio was about two (Mansfeld et al., 1993) while we have constantly observed at least five-fold reading differences. When Pin17/24 was bound to avidin-plates first and then carried out the digestion, it was found that the signal/noise ratio drastically decreased (data not shown). This observation *was consistent with the notion that accessibility of the substrate to the protease is important for the solid-phase-based measurement. Sarubbi e.t al. (1991) also employed ELISA using another recombinant protein substrate, a P-galactosidase-gag fusion protein, to assay protease activity. Again, the fusion protein must be purified from the inclusion bodies and an additional refolding of the recombinant protein is needed for the HIV protease digestion. The method of using crude preparations for detection reagents provides readily an alternative approach for convenient monitoring of a large number of HIV protease reactions.
Acknowledgements We thank T.H. Lee for reagents and continuous encouragement and J.S. Lo for valuable discussion. The work was supported in part by Grant NSC81-0410-BOlO-513 from National Science Council, Taiwan, R.O.C. and an award from Medical Research and Advancement Foundation in Memory of Dr. Chi-Shuen Tsou.
References Billich, S., Knoop. M.-T., Hansen, J., Strop, P., Sedlacek. J., Mertz, R. and Moelling, peptides as substrates and inhibitors of human immune deficiency virus-1 protease. 17905-17908.
K. (1988) Synthetic J. Biol. Chem. 263,
72
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
Boutelje, J., Karlstrom, A.R., Hartmanis, M.G.N., Holmgren, E., Sjogren, A. and Levine, R.L. (1990) Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in E. coli. Arch. Biochem. Biophys. 283, 141-149. Broadhurst, A.V., Roberts, N.A., Ritchie, A.J., Handa, B.K. and Kay, C. (1991) Assay of HIV-1 proteinase: a calorimetric method using small peptide substrates. Anal. Biochem. 193, 280-286. Cheng, Y.S.E., Mcgowan, M.H., Kettner, CA., Schloss, J.V., Viitanen, S.E. and Yin, F.H. (1990) High-level synthesis of recombinant HIV-I protease and the recovery of active enzyme from inclusion bodies. Gene 87, 243-248. Darke, P.L., Nutt, R.F., Brady, S.F., Garsky, V.M., Ciccarone, T.M., Leu, C.T., Lumma, P.K., Freidinger, R.M., Veber, D.F. and Sigal, IS. (1899) HIV-I protease specificity of peptide cleavage is sufftcient for processing of gag and pal polyproteins. Biochem. Biophys. Res. Commun. 155, 297-303. DiIanni, C.L., Davis, L.J., HolIoway, M.K., Herber, W.K., Darke, P.L., Kohl, N.E. and Dixon, R.A.F. (1990) Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease. J. Biol. Chem. 265, 17348-17354. C&am, C.Z. and Boros, I. (1988) In vivo and in vitro autoprocessing of human immunodeficiency virus protease expressed in E. cob. J. Biol. Chem. 263, 14617-14620. Grant, S.K., Deckman, LC., Minnich, M.D., Culp, J., Franklin, S., Dreyer, G.B., Tomaszek, T.A., Debouck, C. and Meek, T.D. (1991) Purification and biochemical characterization of recombinant simian immunodeficiency virus protease and comparison to human immunodeficiency virus type 1 protease. Biochemistry 30, 8424-8434. Johnston, MI. and Hoth, D.F. (1993) Present status and future prospects for HIV therapies. Science 260, 1286-1291. Katoh, I., Yasunaga, T., Ikawa, Y. and Yoshinaga, Y. (1987) Inhibition of retrovial protease activity by an aspartyl proteinase inhibitor. Nature 329, 654-656. Kotler, M., Arad, G. and Hughes, S.H. (1992) Human immunodeficiency virus type 1 gag-protease fusion proteins are enzymatically active. J. Virol. 66, 6781-6783. Kotler, M., Katz, R.A., Danho, W., Leis, J. and Skalka, A. M. (1988) Synthetic peptides as substrates and inhibitors of a retroviral protease. Proc. Nat]. Acad. Sci. USA 8.5, 4185-4189. Lam, P.Y.S., Jadhav, P.K., Eyermann, C.J., Hodge, C.N., Ru, Y., Bacheler, L.T., Meek, J.L., Otto, M.J., Rayner, M.M., Wong, Y.N., Chang, C.-H., Weber, P.C., Jackson, D.A., Sharpe, T.R. and Erickson-Viitanen, S. (1994) Rational Design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors. Science 263, 380-384. Larder, B.A., Kellam, P. and Kemp, S.D. (1993) Convergent combination therapy can select viable multidrugresistant HIV-1 in vitro. Nature 365, 451-453. Levy, J. A. (1993) Pathogenesis of human immunodeficiency virus infection. Microbial. Rev. 57, 183-289. Mansfeld, H.-W., Schulz, S., Grutz, G., von Baehr, R. and Ansorge, S. (1993) Detection of inhibition of HIV-l protease activity by an enzyme-linked immunosorbent assay (ELISA). J. Immunol. Methods, 161, 151-155. Matayoshi, E.D., Wang, G.T., Krafft, G.A. and Erickson, J. (1990) Novel fluorogenic substrates for assaying retrovial proteases by resonnance energy transfer. Science 247, 954-958. Otto, M.J., Garber, S., Winslow, D.L., Reid, C.D., Aldrich, P., Jadhav, P.K., Patterson, C.E., Hodge, C.N. and Cheng, Y.S. (1993) In vitro isolation and identification of HIV variants with reduced sensitivity to C-2 symmetrical inhitors of HIV-l protease. Proc. Nat]. Acad. Sci. USA 90, 7543-7547. Overton, H.A., Fujii, Y., Price, I.R. and Jones, I.M. (1989) The protease and gag gene products of the human immunodeficiency virus: authentic cleavage and post-translatinal modification in an insect cell expression system. Virology 170, 107-116. Ratner, L., Haseltine, W., Patarca, R., Livak, K.J., Starcich, B., Josephs, S.F., Doran, E.R., Rafalski, J.A., Whitehorn, E.A., Baumeister, K., Ivanoff, L., Petteway, S.R.Jr., Pearson, M.L., Lautenberger, J.A., Papas, T.S., Ghrayeb, J., Chang, N.T., Gallo, R.C. and Wong-Staal, F. (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277-284. Richards, A.D., Roberts, R., Dunn, B.M., Graves, M.C. and Kay, J. (1989) Effective blocking of HIV-l proteinase activity by characteristic inhibitors of aspartic proteinases. FEBS Lett. 247, 113-117. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY.
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
73
Sarubbi, E., Nolli, M.L., Andronico, F., Stella, S., Saddler, G., Selva, E., Siccardi, A. and Denaro, M. (1991) A high throughput assay for inhibitors of HIV-1 protease. FEBS Lett. 279, 265-269 Seelmeier, S., Schmidt, H., Turk, V. and von der Helm, K. (1988) Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A. Proc. Natl. Acad. Sci. USA 85, 6612-6616. Smith, D.B. and Johnson, KS. (1988) Single-step purification of polypeptides expressed in E. coli as fusions with glutathione S-transferase. Gene 67, 31-40. Syu, W.-J. and Kahan, L. (1991) Detecting immunocomplex formation in sucrose gradients by enzyme immunoassay. Anal. Biochem. 196, 174-177. Wlodawer, A. and Erickson, J.W. (1993) Structure-base inhibitors of HfV-1 protease. Annu. Rev. Biochem. 62, 543-585. Wondrak, E.M., Copeland, T.D., Louis, J.M. and Oroszlan, S. (1990) A solid phase assay for the protease of human immunodeficiency virus. Anal. Biochem. 188, 82-85. Yu, S.-L.,Chou, M.J., Tam, M.F., Lee, T.H. and Syu, W.-J. (1993) Expression and antigenicity of human immunodeficiency virus type-l transmembrane gp41 in insect cells. Biochem. Biophys. Res. Commun. 191, 207-21.3.
Journal of Virological
Methods 53 (1995) 63-73
Assay of HIV-l protease activity by use of crude preparations of enzyme and biotinylated substrate Sung-Liang Yu, Nay Wang, Chiou-Yi Liou, Wan-Jr Syu* Graduate Instifute of Microbiology and Immunology, National Yang-Ming Uniuersity, Shih-Pai, 112, Taipei, Taiwan, ROC Accepted 6 December
1994
Abstract An enzyme immunoassay was developed for monitoring protease reactions of human immunodeficiency virus (HIV). The protease and its substrate, the gag precursor, were generated separately in Escherichia coli. The HIV-l protease was generated with a glutathione-S-transferase expression system and the gag substrate, named Pin17/24, was prepared with a Pinpoint expression system. Pin17/24 consists of an N-terminal peptide, which is biotinylated in E. coli, fused with a C-terminal peptide that contains a protease cleavage site flanked by p17 and p24 segments. Through its biotin in the N-terminal region, Pin17/24 bound to ELISA plates coated with avidin, whereas through its C-terminal region, the same molecule of Pin17/24 could be recognized b’y an anti-p24 monoclonal antibody. When the protease was added to Pin17/24, the p24 fragment was released from the biotinylated fusion protein and could no longer be retained on the avidin plates, and as a result, binding of the anti-p24 monoclonal antibody decreased. The binding was specific and the reaction was inhibited by a known HIV protease inhibitor. Due to the specific interactions between avidin and biotin, monoclonal antibody and antigen, and the HIV protease and1the gag substrate, crude preparations of these reagents can be used readily in the assay. The simplicity and feasibility of this method should be useful for simultaneous monitoring of many enzyme reactions, particularly for screening possible HIV protease inhibitors. Keywords: Human immunodeficiency ferase; Biotin
l
Corresponding
0166-0934/95/$09.50
virus; Protease; ELISA, Inhibitor;
author. Fax 886 2 821 2880. 0 1995 Elsevier Science B.V. All rights reserved
SSDI 0166-0934(94)00177-4
Fusion protein; Glutathione-S-trans-
64
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
1. Introduction Human immunodeficiency virus (HIV) is the primary agent for the acquired immunodeficiency syndrome (Levy, 1993). Like other retroviruses, HIV encodes an aspartic protease that is responsible for the processing of the viral gag and gag-pol polyproteins. A functionally active protease is essential for the replication and infectivity of HIV. The HIV protease therefore has been recognized as one of the viral target enzymes for anti-HIV therapy (Johnston and Hoth, 1993; Wlodawer and Erickson, 1993). Several inhibitors against this enzyme have been developed and possible usage in patients is being evaluated (Johnston and Hoth, 1993; Lam et al., 1994). Since HIV has high mutation rates and since drug resistant mutants emerge within months after receiving reverse transcriptase inhibitors (Larder et al., 19931, the virus may also evolve quickly into mutants that are resistant to protease inhibitors (Johnston and Hoth, 1993; Otto et al., 1993). One way of dealing with the problem of drug resistance is to develop continuously new drugs. An economic and convenient method for measuring the viral enzyme activity should accelerate the discovery of novel anti-HIV reagents. Most methods used to monitor the protease activity can be divided into two categories. One is to use synthetic peptides or peptide analogs that mimic the junction regions in the native substrates, gag and gag-pol polyproteins (Billich et al., 1988; Darke et al., 1989; Boutelje, et al., 1990; Cheng et al., 1990; Matayoshi et al., 1990; Wondrak et al., 1990; Broadhurst et al., 1991; Lam et al., 1994). The peptide cleavage is detected by high performance chromatography or monitored by absorbance changes. In these systems, the proteases used are purified. Furthermore, these approaches may have to be substantiated with natural substrate reactions (Billich et al., 1988; Kotler et al., 1988). The second category of methods is to use the natural gag substrates expressed in E. coli. The substrates and the enzymes used could be obtained directly from crude bacterial lysates. The enzymatic reactions are monitored by Western blotting analysis of the proteolytic products generated and separated from the substrate precursor (Giam and Boros, 1988; Overton et al., 1989; Dilanni et al., 1990; Grant et al., 1991; Kotler et al., 1992). While the specificity of the reaction can be visualized directly on blots, immunoblotting is tedious and the setting is difficult for simultaneous measurements of numerous reactions. A simple and feasible enzyme-linked immunosorbent assay (ELISA) for detecting HIV-l protease activity is described. A purification step is not necessary for the preparations of the protease, the recombinant substrate, and the monoclonal antibody (MAb) used.
2. Materials and methods 2.1. DNA and immunochemical
reagents
All plasmid transformations were carried out in E. coli strain JM109. DNA manipulations were according to the published protocols (Sambrook et al., 1989). Plasmids after ligation and transformation were checked by restriction enzyme mapping and confirmed further by DNA sequencing of the fusion junctions.
S.-L. Yu et al. /Journal of Virological Methods 53 (1995) 63-73
65
Avidin, avidin-horseradish peroxidase (HRP) conjugates, alkaline phosphatase-labeled goat anti-mouse IgG (whole molecule), and HRP-labeled goat anti-mouse IgG (whole molecule) were obtained from Sigma (St Louis, MO). MAb-containing ascites fluids from BALB,/c mice were used directly without further purification. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Hybridoma 183 (Clone H12-5C), producing MAb against HIV-l ~24, from Dr. Bruce Chesebro. 2.2. Generations of the HlV-1 protease and Pin1 7/24
fusion protein
To produce HIV-l protease, a BgZII/ScaI-restricted fragment of proviral HIV-l DNA HXB2 (nucleotides 2096-2753) (Ratner et al., 1985) was ligated to BamHI/SmaI-digested pGEX-1 (Pharmacia, Uppsala, Sweden). The resulting plasmid pGEXpr encodes a pol-encoded fragment fused to the carboxyl terminus of the glutathione S-transferase (GST) from Schistosoma japonicum (Smith and Johnson, 1988). When induced with isopropyl+?-D-thiogalactopyranoside (IPTG), active HIV-l protease was produced by autocatalytic releasing (Giam and Boros, 1988) from the GST fusion protein (Fig. 1A). To express a gag analog as the HIV-l protease substrate, a Hind111 fragment of HXB2 (nucleotides 1085-1712) was cloned into pBluescript II SK+ (Strategene, La Jolla, CA). The resulting plasmid with the right orientation of insert was then digested with BamHI and HincII. A 0.7-kb fragment was isolated and ligated with the BamHI/EcolRV-restricted Pinpoint Xa-1 vector (Promega, Madison, WI). The expression vector so obtained encoded an about 40 kDa recombinant protein, named Pin17/24. Pin17/24 contains an N-terminal peptide, which is biotinylated at a single lysine residue in E. coli (Promega Technical Manual), fused with a C-terminal gag fragment that consists of a protease cleavage site flanked by p17 and p24 segments (Fig. 1B) 2.3. Preparations of protease and substrate Plasmid-transformed E. coli were cultured in LB broth in the presence of ampicillin (50 pg/ml:l and induced with 1 mM IPTG at 37°C for l-3 h when the optical density at 570 nm reached 0.5. Typically, bacteria of 300 ml culture were pelleted and resuspended in 6 ml substrate buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM P-mercaptoethanol) or 6 ml protease buffer (50 mM Na-acetate pH 5.0, 0.35 M NaCl, 5 mM EDTA, 1 mM PMSF, and 10 mM P-rnercaptoethanol). Bacteria were then lysed with French pressure cell press (SLM Instruments, Urbana, IL). After centrifugation at 6000g for 30 min at 4°C to remove cell debris, the supematants were used directly as the sources for the HIV-l protease or the protease substrate. 2.4. Enzymatic reactions Western blotting was carried out using 4 ~1 of the Pin17/24 lysate mixed with 2 ~1 of the protease lysate and incubated for 1 h at 37°C. The reaction was stopped by adding
66
S. -L. Yu et al. /Journal of Virological Methods 53 (I 995) 63-73
Protease (B)
Biotin
7
I_(
*
~24 fragment
Pin1 7124 Protease M.W.66 45 36 29 24
-
20 -
14 Fig. 1. HIV-l protease generation and reactions with recombinant substrate Pin17/24. (A) Diagram for the HIV-1 protease autocatalytically released from the GST fusion protein (see Section 2, Materials and methods). (B) Illustration for the Pin17/24 recombinant protein and the products after HIV-1 protease digestion. The arrow heads indicate the HIV-1 protease cleavage sites; thin lines represent the sequences derived from the fusion vector sequences whereas thick lines represent that derived from HIV-1 coding sequences. The relative location of biotin on Pin17/24 is also labeled. (C) Western blotting analysis of Pin17/24 with and without HIV-l protease treatment; detection agents used were avidin-HRP and anti-p24 MAb, respectively. F labels the full length Pin17/24. After the HIV-1 protease digestion, Pin17/24 should yield two fragments as illustrated in (B). N labels the 17 kDa N-terminal product with the biotin tag that was detected only by avidin-HRP; C labels the 23 kDa C-terminal fragment that was detected only by the anti-p24 MAb. See Section 4 (Discussion) for explanation of additional bands.
6 pl of 2 X sodium dodecyl sulfate (SDS) sample buffer and boiled. Proteins in the mixtures were separated by SDS-containing polyacrylarnide gel electrophoresis, electroblotted to nitrocellulose filters. The blots were blocked with 3% gelatin in TBS (0.1 M Tris, pH 7.5, 0.5 M NaCl) for 1 h, reacted with avid&conjugated horseradish peroxidase (HRP), and developed with 4-chloro-1-naphthol and H,O, as previously
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
67
described (‘Yu et al., 1993). Alternatively, blots were reacted sequentially with the anti-p24 MAb and HRP-labeled goat anti-mouse antibodies. Blots were then developed accordingly. For ELISA, plates were first coated with 100 ~1 avidin (10 pg/ml in 0.1 M Tris, pH 9.0, 0.15 M NaCl) overnight. After washing with TBS, plates were blocked with 3% bovine serum albumin (BSA) in TBS for 1 h. Plates were used immediately or washed three times with TBS, air-dried, and stored at 4°C until use. Protease reactions in triplicates were performed by mixing 4 ~1 of the Pin17/24 lysate with 2 ~1 of appropriately diluted protease lysates for 1 h at 37°C. 94 ~1 of anti-p24 MAb solutions (ascites fluid 1 to 500 diluted in TBS containing 1% BSA) were added to the mixtures. After a brief mixing, the mixtures were transferred immediately to wells of avidin-coated plates. After incubation at room temperature for 1 h, plates were washed 5 times with TBS. Alkaline phosphatase-conjugated goat anti-mouse antibodies (1 pg/ml in 1% BSA-TBS) were added (100 pi/well) and incubated for an additional 1 h. Plates were then washed 5 times with TBS and developed with 4-methylumbelliferyl phosphate (Sigma) (Syu and Kahan, 1991). The relative fluorescence intensity after alkaline phosphatase hydrolysis was measured using CytoFluor 2300 (Millipore Asia, Taiwan). Pepstatin A (Sigma) was dissolved in dimethyl sulfoxide (DMSO) as a 7 mM stock. Appropriat’e concentrations were made by diluting the stock with DMSO. For a control, an equivalent amount of DMSO was added to the protease reaction.
3. Results To demonstrate that Pin17/24 is an appropriate substrate for HIV-l protease, Western blotting analysis was used with both avidin-HRP and the anti-p24 MAb to monitor the proteolytic reactions (Dilarmi et al., 1990). Consistent with the prediction from the fusion protein construct, Pin17/24 (labeled F in Fig. 1C) has an apparent molecular weight of about 40 kDa and was recognized by both avidin-HRP and the anti-p24 MAb. When HIV-l protease was added to the reaction, Pin17/24 was digested and no longer detected by avidin-HRP. However, the anti-p24 MAb still detected the residual Pin17/24 molecules, indicating that a complete digestion had not been reached and that the anti-p24 MAb detection had a slightly higher sensitivity than that of avidin-HRP. After the HIV-l protease reaction, two digestion products should be generated from Pin17/24 as illustrated in Fig. 1B; the biotinylated N-terminal fragment should be detected only by avidin-HRP while the C-terminal fragment is to be detected only by the anti-p24 MAb. As shown in Fig. 1C a newly generated 17 kDa product (labeled N) was detected only by avidin-HRP and another 23 kDa product (labeled C) was detected only by the anti-p24 MAb. Therefore, these two newly generated products may represent the N-terminal fragment and the C-terminal fragment of the protease-digested Pin17/24, respectively. To adapt Western blotting analysis into a convenient ELISA format, the aim was to simplify the reagent preparations. It was reasoned that plate-coated avidin will bind biotinylated recombinant protein expressed in a crude bacterial lysate with high affinity so that any attempt to purify Pin17/24 is unnecessary. Second, since Pin17/24 has a
68
S.-L. Yu et al. /Journal
1 If; >
of Virological Methods 53 (1995) 63-73
Biotinylated substrate (Pin17/24)
Anti-p24 MAb
Protease
Alkaline phosphatase-conjugated goat anti-mouse Ab
Protease inhibitor (pepstatin A)
R r
(A) Buffer only (B) Protease (C) Protease + Inhibitor -
R4
Fig. 2. A schematic drawing of ELISA measurements of protease reactions. HIV-l protease digestron of Pin17/24 was carried out in microtest tubes for 1 h at 37°C. Solutions with the anti-p24 MAb were then added and the whole mixtures were immediately transferred to avidin-coated ELISA plates. Plate-coated avidin bound the biotin tag in the N-terminal region of Pin17/24, which was in turn recognized by the anti-p24 MAb through the C-terminal region; the plate-retained anti-p24 MAb was then enzymatically measured (see Section 2, Materials and methods) by binding of alkaline phosphatase-conjugated goat anti-mouse IgG (A). When the HIV-1 protease cleaves Pin17/24 into the N-terminal biotin-tagged fragment and the C-terminal p24 fragment, the anti-p24 MAb bound to the p24 fragment could no longer be retained on the plates during the next washing (B). In the presence of an HIV-l protease inhibitor, binding of the anti-p24 MAb to the plates is retained CC).
single biotin in the N-terminal region and a p24 segment in the C-terminal region, simultaneous binding of avidin and the anti-p24 MAb may be possible. Third, a high signal/ noise ratio is highly preferable in the assays. Prior binding of Pin17/24 to plates could restrict the molecular freedom and decrease the accessibility of P17/24 to the HIV-l protease. As a result, the signal/noise ratio of the detection may decrease. To avoid this possibility, the solutions were mixed and the digestion was carried out in microtest tubes. After 1 h digestion, the anti-p24 MAb was added to the tubes and the whole mixtures were transferred to avidin-coated plates. Thereafter, reactions were carried out in the ELISA plates. As illustrated in Fig. 2, when HIV-l protease is functioning, the relative ELBA readings should decrease drastically. When HIV-l protease is inhibited, the ELISA readings are expected to be comparable to that of the buffer controls. Typical results are shown in Fig. 3. As reflected by fluorescence intensity generated from the reaction of alkaline phosphatase bound in the immunocomplexes, the anti-p24 MAb bound on plates decreased progressively as more protease lysates were added. The anti-p24 MAb reached base-line binding after sufficient protease was added to cleave all
S.-L. Yu et al. /Journal
+
7
L -0
c
of Virological Methods 53 (1995) 63-73
loo
GST lysate Pmtease
z m -
69
lysate 0
\ h
_
“i 0 \
‘\ i %.. ‘k.‘.*_......0 ..............”.......* .....“.”..........“.O
0
1
4 Bacteial
Lysate *tied
(~1)
Fig. 3. Dose-dependent cleavage of Pin17/24. Increasing amounts of bacterial lysates containing the HIV-l protease or the control GST molecule were incubated with the Pin17/24 bacterial lysate. Proteolytic cleavage was determined by the decrease of the plate-bound anti-p24 h4Ab (see Section 2, Materials and Methods). Each data point represents a mean of triplicate; standard deviations of the readings were about 10%. Repeated experiments gave similar results.
the digestible Pin17/24. In contrast, the control GST lysate, which derived from the pGEX-1 vector-transformed bacteria, did not decrease efficiently the binding of the anti-p24 even at high concentrations. To substantiate further that the ELISA results did reflect the specific cleavage of HIV-l protease, reactions in the presence of a known inhibitor, pepstatin A (Katoh et al., 1987; Seehneier et al., 1988; Richards et al., 1989), were carried out. Western blotting analysis and ELISA were carried out for a direct comparison. When concentrations of pepstatin A were increased, the cleavage of Pin17/24 and the production of 23 kDa product were suppressed increasingly, as shown clearly in the Western blot (Fig. 4A). Consistent with the Western blotting results, ELISA data (Fig. 4B) indicated that the degrees of inhibition increased accordingly. Pepstatin A at a concentration of 200 PM gave 95% .inhibition, which was comparable to the results of others (Katoh et al., 1987; Seelmeier et al., 1988; Richards et al., 1989).
4. Discussiion A rapid and easy assay was developed to monitor the HIV protease activity. This system has a unique property of using crude preparations of the HIV protease, the recombinant substrate, and the anti-p24 MAb. No purification such as ammonium sulfate precipitation, urea extraction, or refolding is necessary. While Western blotting results are good for qualitative assessment, it is tedious to assay quantitatively the degree of inhibition. Moreover, it is difficult to use Western blotting for analyzing dozens of simultanealus reactions. The ELISA method provided results comparable to that of Western blotting analysis as shown in Fig. 4. Furthermore, ELISA allows quantitative measurements of hundreds of simultaneous protease reactions.
70
S.-L. Yu et al. /Journal
of Virological Methods 53 (I 995) 63- 73
Pin17124 + Protease Pepstatin A
0
0
+ +
+ +
+ +
0
10
50
lb
50
+ + 200 WV
200
Pepstatin A (PM) Fig. 4. Inhibition of HIV-l protease activity analyzed by (A) Western blotting and (B) ELISA. Increasing concentrations of pepstatin A were used to inhibit the HIV-1 protease digestion of Pin17/24. Quaternate digestions were conducted; one sample was analyzed with Western blotting and triplicate were used for ELISA analysis. Western blotting was analyzed with the anti-p24 h4Ab as in Fig. 1 while ELISA was done as in Fig. 3. Standard deviations of the ELISA measurements were about 10%. See legend to Fig. 1 for the explanation of labels F and C.
When recombinant proteins are overproduced in E. coli, nonspecific protease degradation frequently occurs. This was also observed in the preparation of Pin17/24. In the blots with crude bacterial lysates, avidin-HRP detected a 20.5 kDa peptide and the anti-p24 MAb detected a 25 kDa peptide in addition to Pin17/24 (Fig. 1C). Also shown in Fig. lC, both peptides were digested by the HIV-l protease, indicating that both fragments were degraded products of Pin17/24 and all retained the HIV-l protease cleavage site. These degradation products and another minor 18 kDa biotinylated
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
71
product, which was not affected by HIV-l protease (Fig. lC), did not interfere with the ELISA measurement. When the cell lysates of HIV-l-infected CEM cells were used as the source of the protease, the enzyme activity could also be monitored. However, this system was sensitive to SDS and deoxycholate (data not shown). As a result, the HIV-infected cells are normally lysed with the protease buffer containing additional 1% Triton X-100. Recently, Mansfeld et al. (1993) described an ELISA using a recombinant protein as the protease substrate. Their method relies on extracting the recombinant proteins from inclusion bodies and refolding it into digestible substrates. The extraction and refolding are time-consuming and difficult to control for the degree of correct folding. In contrast, our Pin17/24 was soluble in the bacterial lysate and protease digestible without any treatment. Pin17/24 was expressed equally well in E. coli either in the presence of IPTG (Fig. 1C) or without this inducer (data not shown). The ‘leakage’ expression of Pin17/24 offers an additional cost-effective advantage to this substrate preparation. An additional difference between this method and that of Mansfeld et al. (1993) is that we have proteolytic reaction occurred in solutions whereas the latter coat the refolded substrate directly onto plates for protease digestion. Since the steric hindrance of the plate cannot be completely excluded, a portion of the substrates coated on plates may not be accessible to the protease. These inaccessible substrates and molecules with incorrect folding may contribute to a high background binding of the Mab and decrease the signal/noise ratio. Indeed their best signal/noise ratio was about two (Mansfeld et al., 1993) while we have constantly observed at least five-fold reading differences. When Pin17/24 was bound to avidin-plates first and then carried out the digestion, it was found that the signal/noise ratio drastically decreased (data not shown). This observation *was consistent with the notion that accessibility of the substrate to the protease is important for the solid-phase-based measurement. Sarubbi e.t al. (1991) also employed ELISA using another recombinant protein substrate, a P-galactosidase-gag fusion protein, to assay protease activity. Again, the fusion protein must be purified from the inclusion bodies and an additional refolding of the recombinant protein is needed for the HIV protease digestion. The method of using crude preparations for detection reagents provides readily an alternative approach for convenient monitoring of a large number of HIV protease reactions.
Acknowledgements We thank T.H. Lee for reagents and continuous encouragement and J.S. Lo for valuable discussion. The work was supported in part by Grant NSC81-0410-BOlO-513 from National Science Council, Taiwan, R.O.C. and an award from Medical Research and Advancement Foundation in Memory of Dr. Chi-Shuen Tsou.
References Billich, S., Knoop. M.-T., Hansen, J., Strop, P., Sedlacek. J., Mertz, R. and Moelling, peptides as substrates and inhibitors of human immune deficiency virus-1 protease. 17905-17908.
K. (1988) Synthetic J. Biol. Chem. 263,
72
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
Boutelje, J., Karlstrom, A.R., Hartmanis, M.G.N., Holmgren, E., Sjogren, A. and Levine, R.L. (1990) Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in E. coli. Arch. Biochem. Biophys. 283, 141-149. Broadhurst, A.V., Roberts, N.A., Ritchie, A.J., Handa, B.K. and Kay, C. (1991) Assay of HIV-1 proteinase: a calorimetric method using small peptide substrates. Anal. Biochem. 193, 280-286. Cheng, Y.S.E., Mcgowan, M.H., Kettner, CA., Schloss, J.V., Viitanen, S.E. and Yin, F.H. (1990) High-level synthesis of recombinant HIV-I protease and the recovery of active enzyme from inclusion bodies. Gene 87, 243-248. Darke, P.L., Nutt, R.F., Brady, S.F., Garsky, V.M., Ciccarone, T.M., Leu, C.T., Lumma, P.K., Freidinger, R.M., Veber, D.F. and Sigal, IS. (1899) HIV-I protease specificity of peptide cleavage is sufftcient for processing of gag and pal polyproteins. Biochem. Biophys. Res. Commun. 155, 297-303. DiIanni, C.L., Davis, L.J., HolIoway, M.K., Herber, W.K., Darke, P.L., Kohl, N.E. and Dixon, R.A.F. (1990) Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease. J. Biol. Chem. 265, 17348-17354. C&am, C.Z. and Boros, I. (1988) In vivo and in vitro autoprocessing of human immunodeficiency virus protease expressed in E. cob. J. Biol. Chem. 263, 14617-14620. Grant, S.K., Deckman, LC., Minnich, M.D., Culp, J., Franklin, S., Dreyer, G.B., Tomaszek, T.A., Debouck, C. and Meek, T.D. (1991) Purification and biochemical characterization of recombinant simian immunodeficiency virus protease and comparison to human immunodeficiency virus type 1 protease. Biochemistry 30, 8424-8434. Johnston, MI. and Hoth, D.F. (1993) Present status and future prospects for HIV therapies. Science 260, 1286-1291. Katoh, I., Yasunaga, T., Ikawa, Y. and Yoshinaga, Y. (1987) Inhibition of retrovial protease activity by an aspartyl proteinase inhibitor. Nature 329, 654-656. Kotler, M., Arad, G. and Hughes, S.H. (1992) Human immunodeficiency virus type 1 gag-protease fusion proteins are enzymatically active. J. Virol. 66, 6781-6783. Kotler, M., Katz, R.A., Danho, W., Leis, J. and Skalka, A. M. (1988) Synthetic peptides as substrates and inhibitors of a retroviral protease. Proc. Nat]. Acad. Sci. USA 8.5, 4185-4189. Lam, P.Y.S., Jadhav, P.K., Eyermann, C.J., Hodge, C.N., Ru, Y., Bacheler, L.T., Meek, J.L., Otto, M.J., Rayner, M.M., Wong, Y.N., Chang, C.-H., Weber, P.C., Jackson, D.A., Sharpe, T.R. and Erickson-Viitanen, S. (1994) Rational Design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors. Science 263, 380-384. Larder, B.A., Kellam, P. and Kemp, S.D. (1993) Convergent combination therapy can select viable multidrugresistant HIV-1 in vitro. Nature 365, 451-453. Levy, J. A. (1993) Pathogenesis of human immunodeficiency virus infection. Microbial. Rev. 57, 183-289. Mansfeld, H.-W., Schulz, S., Grutz, G., von Baehr, R. and Ansorge, S. (1993) Detection of inhibition of HIV-l protease activity by an enzyme-linked immunosorbent assay (ELISA). J. Immunol. Methods, 161, 151-155. Matayoshi, E.D., Wang, G.T., Krafft, G.A. and Erickson, J. (1990) Novel fluorogenic substrates for assaying retrovial proteases by resonnance energy transfer. Science 247, 954-958. Otto, M.J., Garber, S., Winslow, D.L., Reid, C.D., Aldrich, P., Jadhav, P.K., Patterson, C.E., Hodge, C.N. and Cheng, Y.S. (1993) In vitro isolation and identification of HIV variants with reduced sensitivity to C-2 symmetrical inhitors of HIV-l protease. Proc. Nat]. Acad. Sci. USA 90, 7543-7547. Overton, H.A., Fujii, Y., Price, I.R. and Jones, I.M. (1989) The protease and gag gene products of the human immunodeficiency virus: authentic cleavage and post-translatinal modification in an insect cell expression system. Virology 170, 107-116. Ratner, L., Haseltine, W., Patarca, R., Livak, K.J., Starcich, B., Josephs, S.F., Doran, E.R., Rafalski, J.A., Whitehorn, E.A., Baumeister, K., Ivanoff, L., Petteway, S.R.Jr., Pearson, M.L., Lautenberger, J.A., Papas, T.S., Ghrayeb, J., Chang, N.T., Gallo, R.C. and Wong-Staal, F. (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277-284. Richards, A.D., Roberts, R., Dunn, B.M., Graves, M.C. and Kay, J. (1989) Effective blocking of HIV-l proteinase activity by characteristic inhibitors of aspartic proteinases. FEBS Lett. 247, 113-117. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY.
S.-L. Yu et al. /Journal
of Virological Methods 53 (1995) 63-73
73
Sarubbi, E., Nolli, M.L., Andronico, F., Stella, S., Saddler, G., Selva, E., Siccardi, A. and Denaro, M. (1991) A high throughput assay for inhibitors of HIV-1 protease. FEBS Lett. 279, 265-269 Seelmeier, S., Schmidt, H., Turk, V. and von der Helm, K. (1988) Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A. Proc. Natl. Acad. Sci. USA 85, 6612-6616. Smith, D.B. and Johnson, KS. (1988) Single-step purification of polypeptides expressed in E. coli as fusions with glutathione S-transferase. Gene 67, 31-40. Syu, W.-J. and Kahan, L. (1991) Detecting immunocomplex formation in sucrose gradients by enzyme immunoassay. Anal. Biochem. 196, 174-177. Wlodawer, A. and Erickson, J.W. (1993) Structure-base inhibitors of HfV-1 protease. Annu. Rev. Biochem. 62, 543-585. Wondrak, E.M., Copeland, T.D., Louis, J.M. and Oroszlan, S. (1990) A solid phase assay for the protease of human immunodeficiency virus. Anal. Biochem. 188, 82-85. Yu, S.-L.,Chou, M.J., Tam, M.F., Lee, T.H. and Syu, W.-J. (1993) Expression and antigenicity of human immunodeficiency virus type-l transmembrane gp41 in insect cells. Biochem. Biophys. Res. Commun. 191, 207-21.3.