Journal of Biotechnology, 21 (1991) 127-136
127
© 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1656/91/$03.50 ADONIS 016816569100148X BIOTEC 00680
Large scale expression and purification of recombinant HIV-1 proteinase from Escherichia coli O n k a r M.P. Singh l, D e v S. B a i n e s z, R i c h a r d M. Hall z, N o r m a n M. G r a y 3 and M a l c o l m P. W e i r 1 Departments of i Genetics, 2 Natural Products Discovery and ~ Virology, Glaxo Group Researcl, Ltd., Greenford, Middlesex, U.K.
(Received 14 February 1991; revision accepted 28 May 1991)
Summary The availability of target proteins in sufficient quantity is a limiting factor in crystallographic studies and therefore in rational drug design. Even after optimisation, expression of recombinant proteins may be low and the only way to produce enough protein is by large scale cell growth/purification. HIV-1 proteinase in Escherichia coli, which due to its toxicity is expressed as a soluble protein only at around 0.1% of total protein, is a paradigm for this. In this paper a detailed process for large scale expression and purification of HIV-1 proteinase which delivers material of suitable quantity (30 mg from 500 g of wet weight of cells) and quality for crystallographic studies is described. HIV proteinase; Scale-up; Escherichia coli; Recombinant protein
Introduction HIV-1 proteinase (HIVP) is a primary target for chemotherapy of AIDS and HIV infection; it is responsible for the cleavage of gag and gag-pol fusion proteins and inhibition of this processing results in immature particles which are rendered non-infectious (Kohl et al., 1988; McQuade et al., 1990); in vivo inhibition of HIVP should thus reduce the spread of virus both from lysed and chronically infected Correspondence to: M. Weir, Dept. of Genetics, Glaxo Group Research Ltd., Greenford Road,
Greenford, Middlesex UB6 0HE, U.K.
128
cells. HIVP is a symmetrical dimer of 99-residue subunits which are self-cleaved from the gag-pol fusion protein and it has been shown to be an aspartic proteinase (Pearl and Taylor, 1987; Wlodawer et al., 1989). Its dimeric nature is thought to be unique to retroviral aspartic proteinases and crystal structures of synthetic or E. coli expressed HIVP containing bound peptoid inhibitors show many features distinct from mammalian aspartic proteinases (Miller et al., 1989), leading to considerable specificity of binding (Erickson et al., 1990). Structural information on HIVP is proving to be of considerable value in inhibitor design, and requires a steady supply (tens of milligrams per month) of highly purified active protein for crystallography and NMR spectroscopy. HIVP presented several difficulties in this regard; it is highly toxic to ceils, and can generally only be expressed in soluble form in small quantities (Darke et al., 1989); it also tends to be inactivated during purification probably due to oxidation of a cysteine involved in dimerisation and to autoproteolysis (Strickler et al., 1989). Scale-up therefore, reqiaires careful optimisation of growth conditions and purification steps. Several small-scale, medium scale or outline purification methods have been published (Darke et al., 1989; Hansen et al., 1988; McKeever et al., 1989; Danley et al., 1989); we present here a detailed description of the scale-up of HIVP expression and purification for structural studies, which illustrates that the preparation of quantities of protein suitable for crystallography from a system in which expression levels are very low is feasible. Materials and Methods
Materials Chemicals were from BDH, Poole, U.K. Substrate peptide for the protease assay was from Protein and Peptide Research Consultants (University of Reading, U.K.). Chromatography matrices were from Pharmacia LKB Biotechnology (Milton Keynes, U.K.). Bacterial strain and expression vector The host strain employed was E. coli RB791 obtained from the E. coli Genetic Stock Centre, Yale University, School of Medicine, U.S.A. Complete details of the plasmid construct (pTCEMatP1) have been previously reported (Montgomery et al., 1991). Essentially, a NcoI-SalI fragment encoding residues 1-167 of the POL open reading frame (including the proteinase auto-processing site Phe68-Pro69) was fused to the chloramphenicol acetyl transferase gene, which is expressed under the control of the Tac promoter. The resulting vector encoded a 27 kDa fusion protein with autocleavage releasing the mature 11 kDa proteinase. Methods Conditions for cell growth and expression Large scale production was carried out in a Biolaffitte bioreactor batched to a working volume of 450 I. Complete details of the optimised process are shown in
129 Scale
E. coil RB791 (pTCE Mat Pl) (freeze dried culture)
I
M3 plate (37°C; 24 h)
I
50 ml
M3 Medium (37°C; 250 rpm; 8 h)
4 I
M3 Medium (37°C; 750 rpm; *4 Ipm; 8 h)
450 I
M3 Medium (37°C; 350 rpm; 450 Ipm; 9 h
I
I
I
Cell harvest with rapid cooling (typically 3-5 kg wet wt.)
Fig. 1. Summary of optimised large scale process for growth and expression of HIV-1 proteinase. Ampicillin was added at all stages at 50-100/zg ml-1 as a filter sterilised solution. Inducer (IPTG) was added after 6 h to the 4501 culture with cells harvested 3 h later. * lpm: aeration in 1min- i. Fig. 1. A Rushton impeller was used. The growth medium (M3) consisted of glucose 20 g 1-1 , casamino acids 20 g l - I , K 2 H P O 4 7 g 1- l , K H 2 P O 4 3 g 1-x, (NH4)2SO 4 1 g 1-1, N a 3 C 6 H s O 7 . 2 H 2 0 0.5 g l - l , M g S O 4 - 7 H 2 0 0.5 g l - l , thiamine HCI 2 mg 1-~ with ampicillin 100 mg 1-l added to maintain plasmid. The antifoam agent PPG2000 was routinely added at a concentration of 1.5 ml I - l to reactors, p H control was not found to be necessary for optimal growth or proteinase expression. (Growth conditions for both shake flask and 4 1 scale cultures are also shown in Fig. 1). The inducer, isopropyl /3-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 m M to the production vessel following growth for 6 h. Cells were harvested to coincide with peak proteinase activity which was typically 3 h after induction. Rapid cooling of the vessel to approx. 18 ° C was carried out prior to harvesting cells.
Cell harvesting and extraction Cells from a 450 1 culture were harvested using a Sharpies AS16 continuous flow centrifuge, aliquoted into 500 g batches and stored at - 7 0 ° C. One aliquot was thawed at 4 o C mixed into 2 1 of buffer containing 50 mM MES, 2 m M E D T A , 1 mM D T F and 0.01% Triton X-100, p H 6.0 (buffer A) and passed three times through a Manton-Gaulin homogeniser (8000 psi, 4 o C). The cell lysate was slowly dripped into 10 volumes of acetone, allowed to precipitate for 30 min and then filtered through Whatman-54 filter paper. The semi dried pellet was then extracted with buffer A containing 0.1% Triton X-100 0.5 M NaCI and 50% glycerol. This extract can be prepared 24 h in advance of the chromatography and stored at - 2 0 o C.
Enzyme assay Enzyme activity was determined by following the cleavage of a peptide, derived from C-terminus of the proteinase and the N-terminus of reverse transcriptase, with the sequence K Q G T V S F N F P Q I T . The conditions of the assay and the separation of the fragments were as described (Singh et al., 1990) except that a Spherisorb ODS2 3 / z m reversed phase H P L C column (Anachem) was used and developed isocratically with 27% acetonitrile.
130
Chromatography The soluble extract was diluted 10-fold at the head of the S-Sepharose Fast Flow anion exchange (IEC) column (100 c m x 7 cm) by delivering 10% extract and 90% buffer A using a Pharmacia Biopilot system. The flow rate for the loading was 80 ml rain- ~. After the sample was loaded the matrix was washed with 1 1 of buffer A followed by a reverse wash with 2 1 of buffer without Triton X-100. Bound proteinase was eluted with a 0-1.0 M NaCI gradient in buffer A followed by a further 1 I wash with buffer containing 1 M NaCI. 50 ml fractions were collected on ice. Pooled fractions containing the enzyme were then loaded onto an Alkyl-Superose column (2.6 cm × 7 cm) by a similar mixing procedure as for ion-exchange. 50% dilution with 3.4 M ammonium sulphate in 25 mM Citrate, 2 mM EDTA, 1 mM D ' I T (buffer B), pH 6.0 at a loading rate of 12 ml m i n - 1. The matrix was then washed with 500 ml buffer B containing 1.6 M ammonium sulphate and the bound enzyme was eluted in reverse flow by isocratic elution with 220 ml buffer A at a flow rate of 4.0 ml m i n - L NaCI and glycerol were added to the recovered protein to obtain 2 M and 10% final concentration respectively and then the sample was concentrated under nitrogen using Amicon YM10 membrane containing concentrator, to a volume of 40 ml. The concentrated material was centrifuged, at 10,000 x g for 30 min, to remove precipitated proteins. The soluble supernatant liquid was loaded on to a 1.8 1 Superdex-G75 column (5 cm x 95 era) which had been equilibrated with 50 mM MES, 2 mM EDTA, 1 mM D T r , 10% glycerol, 0.5 M NaCI buffer at pH 6.0. The loading flow rate was 2 ml min-1 and once loaded this was increased stepwise to 10 ml min-1. Fractions of 10 ml were collected on ice.
Protein determination Protein content of the crude and partially purified material was determined by the Pierce Coomassie Blue dye binding method (Pierce and Warriner Ltd.) and the concentration of purified protein was determined by using the calculated extinction coefficient of 1.14 for a 1 mg m l - l solution at 280 nm. SDS-polyacrylamide gel electrophoresis separations and silver staining was as previously described (Montgomery et al., 1991).
Fluorescence spectroscopy Steady state fluorescence spectra of HIV-1 proteinase were recorded on a Perkin Elmer LS5B spectrofluorimeter with 2.5 nm excitation and 5 nm emission slit widths, 285 nm excitation wavelength. The proteinase concentration was 40/~g ml-1 in 50 mM MES pH 6, 0.5 M NaC1, 2 mM EDTA, 35% glycerol. Results and Discussion
Cell growth and expression HIV-proteinase expression was initially optimised using shake flask and 4 1 cultivations before scale up into the production vessel. Expression had been
131
12 •
.
L
lOo
0
86-
42
0 4 8
1'2 16 2'0 24 h
120 Q)
B
' 1'27::
~100
t 1'0 {:
'~ =e 80 .o 60 '6 :.5 40
.,%\ / \\t L
20 o
,
0
4
_
,
,
. . . . . . . .
,
~o.8~
~o
8 12 16 20 24
e
h u.I
Fig. 2. Expression of HIVP at 4 1 scale. The growth medium was M3, temperature 37 ° C, agitation 750 rpm, aeration 4 1 min-L. A. Comparison of cell growth (OD550) in the presence ( • ) and absence (o) of inducer. B. Decrease in antibiotic resistance following overnight growth ( • ) and protease activity following induction (o) showing a decline in proteinase activity with an extended growth period.
previously reported to have a detrimental effect on the host strain (Darke et al., 1989) and this was confirmed under our conditions (Fig. 2A). Following these initial experiments it was clear that cell growth needed to be maximised prior to induction since little increase in biomass was observed following addition of the inducer (IPTG). A short cell growth period was also favoured for a number of reasons including: (a) Proteinase activity decayed to zero if the cell growth was allowed to run overnight after induction (see Fig. 2B). (b) Loss of plasmid following overnight growth (see Fig. 2B). (c) Ceils expressing proteinase became increasingly viscous and difficult to process with an extended growth period. (d) As the product was unstable losses would be minimised with a short cell period. Before further scale up the level of IPTG was optimised at the 4 1 scale, the final level being fixed at 1 mM. The optimised process at the 450 1 scale is illustrated by Fig. 3. Typically, at induction (6 h) the ODs50 was 3-7 with cells harvested some 3 h later to coincide with peak proteinase activity. No detectable activity was observed prior to induction. A marked decrease in CO 2 production (a measure of cells undergoing active respiration) coincided with the start of proteinase expression again highlighting the deleterious nature of the expressed product. It is possible that the proteinase uses a key cellular component as a substrate leading to a loss of cellular activity. Production at the 450 1 scale typically yielded 3-5 kg wet wt of cells.
132 .C
!l
7"
~4 ~q
\
=0..t 0. t
•
0
~
0
c:i
-
, / W=io
olo
=°i' °1 =io
=io
s'.o
~o'.o
h
Fig. 3. Expression of HIVP at 450 1 scale. Conditions are defined in Fig. 1. HIVP expression (e) upon induction leads to toxicity as shown by a slowing down in growth ( • ) and a drop in exhaust CO 2 (o).
Purification A small-scale method for production of 50 p.g HIVP was developed before initiation of scale-up (Montgomery et al., 1991), comprising acetone precipitation of crude cell lysate (Hansen et aI., 1988), ion-exchange (IEC), hydrophobic interaction on alkyl Superose (HIC) and gel permeation chromatography (GPC). All of these steps proved to be scaleable from Pharmacia FPLC to BioPilot scale. Acetone precipitation was retained as a first step since it improved the recovery of enzyme, possibly due to release of HIVP loosely associated with lipid. Subsequent extraction of acetone powder (which can be stored for several months at - 7 0 °C without loss of activity) with 0.5 M NaCl/0.1% Triton X-100/40% glycerol pH 6.0 is a critical stage; inadequate homogenising of the acetone powder leads to HIVP remaining aggregated and voiding from the subsequent IEC column. Extracted acetone powder was diluted and immediately loaded onto the ion exchange column followed by reverse elution with an NaCl gradient (Fig. 4A). Pooled active fractions were then made up to 1.6 M ammonium sulphate at the head of the hydrophobic interaction column; 1.6 M ammonium sulphate is close to the salting-out point of HIVP but this concentration was found to be necessary for good retention on HIC. Fractions from the HIC column were eluted into glycerol to preserve activity, concentrated overnight 5-fold and loaded onto Superdex G75 gel filtration (GPC) column pre-equilibrated in 50 mM Hepes, pH 6.0, containing 0.5 M NaC1, 1 mM DTI', 2 mM EDTA (Fig. 4B); proteinase activity elutes at around 11 kDa molecular mass under these salt conditions, which may be due to dissociation to the monomer in the absence of substrate (Singh et al., 1990), although ultracentrifugation evidence suggests a stable dimeric form under similar conditions (Meek et al., 1989). The GPC stage affords a 2.7-fold purification. The overall purification
133 13.2 -
1.2-
--" 11.0-
1.0-
'=_ E
L E "6
8.8.
0.8-
i~ ~ 6.6. '~ 0.6~
4.4-
o.4-
2.2 -
0.2-
O-
2~o
~o
7;,o
lo~o
12'so
1~
17~o
2~
2='so
Elution Vol (ml) 8.~8-
0.06-
_~ 6.66.
0.06-
¢ 4,44-
0.04-
~ 2,22-
0.02 •
B
128 K
44 K
17 K
135 K
:E
O-
0
600
800 1000 Elution Vol (ml)
1200
1400
1600
1800
Fig. 4. (A) Ion exchange chromatography of solubilised acetone powder on S-Sepharose Fast Flow, HIVP activity (o) is eluted by 0.5-1 M NaCI and loaded onto HIC after addition of (NH4)2SO 4. (B) Gel permeation chromatography of concentrated ex HIC pool on Superdex G75, the final step in purification. Active fractions (e) are 90-95% pure HIVP; inhibitors for co-crystallisation are added at this stage. TABLE 1 Purification of HIV-1 proteinase from 500 g wet weight cells
Disrupted cells Acetone powder Ex IEC Ex HIC * Ex GPC
Protein (mg ml - t)
Volume (ml)
Total protein (mg)
18.4
2200
40480
2.84
1800
5107
0.57 0.90 0.039
1420 152 782
812 113 30.7
Sp. Act, (nmoles min - 1) min mg - l )
Total Act. (tzmoles min- l)
Recovered Act. (%)
2243
100
470
2 400
107
2158 10408 27 737
1752 1176 744
78 52 33
55.4
* GPC load was concentrated to 100 ml on an Amicon YM10 and loaded in two 50 ml runs onto GPC.
134 M'W
K -
1
2
3
4
o
.o
•
K
5
Fig. 5. Silver-stained SDS-PAGE of purified HIVP. Tracks 2, 4; molecular weight markers. Tracks 1, 3, 5; 500 rig, 250 ng and 125 ng of HIVP respectively. The major band at 11 kDa is consistent with the expected molecular weight.
20
~ 15fe
-_= 8C 8 G)
_o= u_ 5-
o 300
~ 320
340 360 380 400 Emission wavelength (nm)
420
Fig. 6. Fluorescence spectrum of HIVP, excitation at 285 nm. The emission spectrum is dominated by the two tryptophans which are largely exposed, according to the crystal structure; the emission mmdmum (345 nm) is in agreement with this observation.
135
scheme starting with 500 g cells is summarised in Table 1; in order to get good overall recovery and yield it was essential to keep columns and buffers cold, use MilliQ water, keep glycerol/DTT in the solvent, work as quickly as possible (the whole process could be completed in 2.5 d) and finally, before concentration for structural studies, it is necessary to inhibit the enzyme to prevent autodegradation. The overall yield is, as far as can be ascertained, comparable to previous reports, also from E. coli (McKeever et al., 1989; Danley et al., 1989). Several hundred mg of the HIVP have been obtained by this process. The protein is 90-95% pure HIVP as judged by SDS-PAGE (Fig. 5). N-terminal sequence analysis by Edman degradation confirmed the correct N-terminus and high purity of the preparation. The specific activity is about 75% the value found for HIVP purified on the analytical scale (Montgomery et al., 1991), suggesting some inactivation has occurred during the longer preparative procedure in spite of the precautions taken. Fluorescence spectroscopy (Fig. 6) is another useful monitor of protein integrity; HIVP has two tryptophans, one of which, Trp6, is fully exposed and the other, Trp42, slightly buried according to the crystal structures (Wlodawer et al., 1989; Miller et al., 1989). The fluorescence spectrum is consistent with this structural data; Trp fluorescence decreases in emission wavelength in apolar environments (Campbell and Dwek, 1984). Maximal emission at 345 nm (Fig. 6) is a little below the value of 352 nm expected for fully exposed Trp. A further indication of the quality of the protein comes from its crystallisation. HIVP prepared as described was found to crystallise in 1 M NaCI pH 7.0 from hanging drops; crystals were tetragonal (P41) and diffracted to 2.7/~ at the SERC Daresbury Synchrotron source, and are thus essentially the same as crystals first prepared by McKeever et al. (1989). Co-crystals with proprietary ligands are now being used for diffraction studies.
Acknowledgements Many thanks to Mr. G.M. Turcatti, Glaxo Institute of Molecular Biology, Geneva for N-terminal sequencing, to Miss E. Roud-Mayne and Mr. J. Piercey for help in purification and to Dr. T.J.R. Harris, Dr. A.N. Hobden and Dr. M.J. Dawson for helpful comments.
References Campbell, I.D. and Dwek, R.A. (1984) Fluorescence, Chapter 5. In Biological Spectroscopy, Benjamin, Cummings Publishing Company Inc. Danley, D.E., Geoghegan, K.F., Scheld, K.G., Lee, S.E., Merson, J.R., Hawrylik, S.J., Rickett, G.A., Ammiratic, M.J. and Hobart, P.M. (1989) Crystallisable HIV-1 protease derived from expression of the viral pol gene in Escherichia coll. Biochem. Biophys. Res. Comm. 165, 1043-1050. Darke, P.L., Leu, C-T., Davis, L.J., Heinbach, J.C., Diehl, R.E., Hill, W.S., Dixon, R.A.F. and Sigal, I.S. (1989) HIV protease: bacterial expression and characterisation of the purified aspartic protease. J. Biol. Chem. 264, 2307-2312.
136 Erickson, J., Neidhart, D.J., Van Drie, J., Kempf, D.J., Wang, X.C., Norbeck, D.W., Plattner, J.J., Rittenhouse, J.W., Turon, M., Wideburg, N., Kohlbrenner, W.E., Simmer, R., Helfrich, R., Paul, D.A. and Knigge, M. (1990) Design, activity and 2.8 A crystal structure of C 2 symmetric inhibitor complexed to HIV-1 protease. Science 249, 527-533. Hansen, J., Billich, S., Schulze, T., Sukrow, S. and Moelling, K. (1988) Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody. Eur. Mol. Biol. Org. J. 7, 1785-1791. Kohl, N.E., Emini, E.A., Schlief, W.A., Davis, L.J., Heinbech, J.C., Dixon, R.A.F., Scolnick, E.M. and Sigal, I.S. (1988) Active HIV protease is required for viral infectivity. Proc. Natl. Acad. Sci. U.S.A. 85, 4686-4690. McKeever, B.M., Navia, M., Fitzgerald, P.M.D., Springer, J.P., Leu, C.T., Heinbach, J.C., Herber, W.K., Sigal, I.S. and Darke, P.L. (1989) Crystallisation of the aspartyl-protease from HIV-1. J. Biol. Chem. 264, 1919-1921. McQuade, T.J., Tomasselli, A.G., Liu, L., Karacostas, V., Moss, B., Sawyer, T.K., Henrikson, R.L. and Tarpley, W.G. (1990) A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation. Science 247, 454-456. Meek, T.D., Dayton, B.D., Metcalf, B.W., Dreyer, G.B., Strickler, J.E., Gorniak, J.G., Rosenberg, M., Moore, M.L., Magaard, V.W. and Debouck, C. (1989) HIV-1 protease expressed in E. coli behaves as a dimeric aspastic protease. Proc. Natl. Acad. Sci. U.S.A. 86, 1841-1845. Miller, M., Schneider, J., Sathyanarayanara, B.K., Toth, M.V., Marshall, G.R., Clawson, L., Selk, L., Kent, S.B.H. and Wlodawer, A. (1989) Structure of complex of synthetic HIV protease with a substrate-based inhibitor at 2.3 A resolution. Science 246, 1149-1152. Montgomery, D.S., Singh, O.M.P., Gray, N.M., Dykes, C.W., Weir, M.P. and Hobden, A.N. (1991) Expression of an autoprocessing Cat-HIV1 proteinase fusion protein: purification to homogeneity of the released 99 residue proteinase. Biochem. Biophys. Res. Comm., submitted for publication. Pearl, L.H. and Taylor, W.R. (1987) A structural model for the retroviral proteases. Nature 329, 351-354. Singh, O.M.P., Roud-Mayne, E. and Weir, M.P. (1990) Dimerisation of HIV-1 protease, In: Pearl, L.H., Ed., Retroviral proteases, Maturation and Morphogenesis, Macmillan Press Ltd., N.Y., U.S.A., pp. 73-78. Strickler, J.E., Gorniak, J., Dayton, B., Meek, T., Moore, M., Magaard, V., Malinowski, J. and Debouck, C. (1989) Characterisation and autoprocessing of mature forms of HIV-1 protease from E. coli. Proteins 6, 139-154. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B.K., Baldwin, E., Weber, I.T., Selk, L.M., Clawson, L., Schneider, J. and Kent, S.B.H. (1989) Conserved folding in retroviral proteases: crystal structure of synthetic HIV-1 protease. Science 245, 616-621.
127
© 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1656/91/$03.50 ADONIS 016816569100148X BIOTEC 00680
Large scale expression and purification of recombinant HIV-1 proteinase from Escherichia coli O n k a r M.P. Singh l, D e v S. B a i n e s z, R i c h a r d M. Hall z, N o r m a n M. G r a y 3 and M a l c o l m P. W e i r 1 Departments of i Genetics, 2 Natural Products Discovery and ~ Virology, Glaxo Group Researcl, Ltd., Greenford, Middlesex, U.K.
(Received 14 February 1991; revision accepted 28 May 1991)
Summary The availability of target proteins in sufficient quantity is a limiting factor in crystallographic studies and therefore in rational drug design. Even after optimisation, expression of recombinant proteins may be low and the only way to produce enough protein is by large scale cell growth/purification. HIV-1 proteinase in Escherichia coli, which due to its toxicity is expressed as a soluble protein only at around 0.1% of total protein, is a paradigm for this. In this paper a detailed process for large scale expression and purification of HIV-1 proteinase which delivers material of suitable quantity (30 mg from 500 g of wet weight of cells) and quality for crystallographic studies is described. HIV proteinase; Scale-up; Escherichia coli; Recombinant protein
Introduction HIV-1 proteinase (HIVP) is a primary target for chemotherapy of AIDS and HIV infection; it is responsible for the cleavage of gag and gag-pol fusion proteins and inhibition of this processing results in immature particles which are rendered non-infectious (Kohl et al., 1988; McQuade et al., 1990); in vivo inhibition of HIVP should thus reduce the spread of virus both from lysed and chronically infected Correspondence to: M. Weir, Dept. of Genetics, Glaxo Group Research Ltd., Greenford Road,
Greenford, Middlesex UB6 0HE, U.K.
128
cells. HIVP is a symmetrical dimer of 99-residue subunits which are self-cleaved from the gag-pol fusion protein and it has been shown to be an aspartic proteinase (Pearl and Taylor, 1987; Wlodawer et al., 1989). Its dimeric nature is thought to be unique to retroviral aspartic proteinases and crystal structures of synthetic or E. coli expressed HIVP containing bound peptoid inhibitors show many features distinct from mammalian aspartic proteinases (Miller et al., 1989), leading to considerable specificity of binding (Erickson et al., 1990). Structural information on HIVP is proving to be of considerable value in inhibitor design, and requires a steady supply (tens of milligrams per month) of highly purified active protein for crystallography and NMR spectroscopy. HIVP presented several difficulties in this regard; it is highly toxic to ceils, and can generally only be expressed in soluble form in small quantities (Darke et al., 1989); it also tends to be inactivated during purification probably due to oxidation of a cysteine involved in dimerisation and to autoproteolysis (Strickler et al., 1989). Scale-up therefore, reqiaires careful optimisation of growth conditions and purification steps. Several small-scale, medium scale or outline purification methods have been published (Darke et al., 1989; Hansen et al., 1988; McKeever et al., 1989; Danley et al., 1989); we present here a detailed description of the scale-up of HIVP expression and purification for structural studies, which illustrates that the preparation of quantities of protein suitable for crystallography from a system in which expression levels are very low is feasible. Materials and Methods
Materials Chemicals were from BDH, Poole, U.K. Substrate peptide for the protease assay was from Protein and Peptide Research Consultants (University of Reading, U.K.). Chromatography matrices were from Pharmacia LKB Biotechnology (Milton Keynes, U.K.). Bacterial strain and expression vector The host strain employed was E. coli RB791 obtained from the E. coli Genetic Stock Centre, Yale University, School of Medicine, U.S.A. Complete details of the plasmid construct (pTCEMatP1) have been previously reported (Montgomery et al., 1991). Essentially, a NcoI-SalI fragment encoding residues 1-167 of the POL open reading frame (including the proteinase auto-processing site Phe68-Pro69) was fused to the chloramphenicol acetyl transferase gene, which is expressed under the control of the Tac promoter. The resulting vector encoded a 27 kDa fusion protein with autocleavage releasing the mature 11 kDa proteinase. Methods Conditions for cell growth and expression Large scale production was carried out in a Biolaffitte bioreactor batched to a working volume of 450 I. Complete details of the optimised process are shown in
129 Scale
E. coil RB791 (pTCE Mat Pl) (freeze dried culture)
I
M3 plate (37°C; 24 h)
I
50 ml
M3 Medium (37°C; 250 rpm; 8 h)
4 I
M3 Medium (37°C; 750 rpm; *4 Ipm; 8 h)
450 I
M3 Medium (37°C; 350 rpm; 450 Ipm; 9 h
I
I
I
Cell harvest with rapid cooling (typically 3-5 kg wet wt.)
Fig. 1. Summary of optimised large scale process for growth and expression of HIV-1 proteinase. Ampicillin was added at all stages at 50-100/zg ml-1 as a filter sterilised solution. Inducer (IPTG) was added after 6 h to the 4501 culture with cells harvested 3 h later. * lpm: aeration in 1min- i. Fig. 1. A Rushton impeller was used. The growth medium (M3) consisted of glucose 20 g 1-1 , casamino acids 20 g l - I , K 2 H P O 4 7 g 1- l , K H 2 P O 4 3 g 1-x, (NH4)2SO 4 1 g 1-1, N a 3 C 6 H s O 7 . 2 H 2 0 0.5 g l - l , M g S O 4 - 7 H 2 0 0.5 g l - l , thiamine HCI 2 mg 1-~ with ampicillin 100 mg 1-l added to maintain plasmid. The antifoam agent PPG2000 was routinely added at a concentration of 1.5 ml I - l to reactors, p H control was not found to be necessary for optimal growth or proteinase expression. (Growth conditions for both shake flask and 4 1 scale cultures are also shown in Fig. 1). The inducer, isopropyl /3-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 m M to the production vessel following growth for 6 h. Cells were harvested to coincide with peak proteinase activity which was typically 3 h after induction. Rapid cooling of the vessel to approx. 18 ° C was carried out prior to harvesting cells.
Cell harvesting and extraction Cells from a 450 1 culture were harvested using a Sharpies AS16 continuous flow centrifuge, aliquoted into 500 g batches and stored at - 7 0 ° C. One aliquot was thawed at 4 o C mixed into 2 1 of buffer containing 50 mM MES, 2 m M E D T A , 1 mM D T F and 0.01% Triton X-100, p H 6.0 (buffer A) and passed three times through a Manton-Gaulin homogeniser (8000 psi, 4 o C). The cell lysate was slowly dripped into 10 volumes of acetone, allowed to precipitate for 30 min and then filtered through Whatman-54 filter paper. The semi dried pellet was then extracted with buffer A containing 0.1% Triton X-100 0.5 M NaCI and 50% glycerol. This extract can be prepared 24 h in advance of the chromatography and stored at - 2 0 o C.
Enzyme assay Enzyme activity was determined by following the cleavage of a peptide, derived from C-terminus of the proteinase and the N-terminus of reverse transcriptase, with the sequence K Q G T V S F N F P Q I T . The conditions of the assay and the separation of the fragments were as described (Singh et al., 1990) except that a Spherisorb ODS2 3 / z m reversed phase H P L C column (Anachem) was used and developed isocratically with 27% acetonitrile.
130
Chromatography The soluble extract was diluted 10-fold at the head of the S-Sepharose Fast Flow anion exchange (IEC) column (100 c m x 7 cm) by delivering 10% extract and 90% buffer A using a Pharmacia Biopilot system. The flow rate for the loading was 80 ml rain- ~. After the sample was loaded the matrix was washed with 1 1 of buffer A followed by a reverse wash with 2 1 of buffer without Triton X-100. Bound proteinase was eluted with a 0-1.0 M NaCI gradient in buffer A followed by a further 1 I wash with buffer containing 1 M NaCI. 50 ml fractions were collected on ice. Pooled fractions containing the enzyme were then loaded onto an Alkyl-Superose column (2.6 cm × 7 cm) by a similar mixing procedure as for ion-exchange. 50% dilution with 3.4 M ammonium sulphate in 25 mM Citrate, 2 mM EDTA, 1 mM D ' I T (buffer B), pH 6.0 at a loading rate of 12 ml m i n - 1. The matrix was then washed with 500 ml buffer B containing 1.6 M ammonium sulphate and the bound enzyme was eluted in reverse flow by isocratic elution with 220 ml buffer A at a flow rate of 4.0 ml m i n - L NaCI and glycerol were added to the recovered protein to obtain 2 M and 10% final concentration respectively and then the sample was concentrated under nitrogen using Amicon YM10 membrane containing concentrator, to a volume of 40 ml. The concentrated material was centrifuged, at 10,000 x g for 30 min, to remove precipitated proteins. The soluble supernatant liquid was loaded on to a 1.8 1 Superdex-G75 column (5 cm x 95 era) which had been equilibrated with 50 mM MES, 2 mM EDTA, 1 mM D T r , 10% glycerol, 0.5 M NaCI buffer at pH 6.0. The loading flow rate was 2 ml min-1 and once loaded this was increased stepwise to 10 ml min-1. Fractions of 10 ml were collected on ice.
Protein determination Protein content of the crude and partially purified material was determined by the Pierce Coomassie Blue dye binding method (Pierce and Warriner Ltd.) and the concentration of purified protein was determined by using the calculated extinction coefficient of 1.14 for a 1 mg m l - l solution at 280 nm. SDS-polyacrylamide gel electrophoresis separations and silver staining was as previously described (Montgomery et al., 1991).
Fluorescence spectroscopy Steady state fluorescence spectra of HIV-1 proteinase were recorded on a Perkin Elmer LS5B spectrofluorimeter with 2.5 nm excitation and 5 nm emission slit widths, 285 nm excitation wavelength. The proteinase concentration was 40/~g ml-1 in 50 mM MES pH 6, 0.5 M NaC1, 2 mM EDTA, 35% glycerol. Results and Discussion
Cell growth and expression HIV-proteinase expression was initially optimised using shake flask and 4 1 cultivations before scale up into the production vessel. Expression had been
131
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.
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,
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,
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Fig. 2. Expression of HIVP at 4 1 scale. The growth medium was M3, temperature 37 ° C, agitation 750 rpm, aeration 4 1 min-L. A. Comparison of cell growth (OD550) in the presence ( • ) and absence (o) of inducer. B. Decrease in antibiotic resistance following overnight growth ( • ) and protease activity following induction (o) showing a decline in proteinase activity with an extended growth period.
previously reported to have a detrimental effect on the host strain (Darke et al., 1989) and this was confirmed under our conditions (Fig. 2A). Following these initial experiments it was clear that cell growth needed to be maximised prior to induction since little increase in biomass was observed following addition of the inducer (IPTG). A short cell growth period was also favoured for a number of reasons including: (a) Proteinase activity decayed to zero if the cell growth was allowed to run overnight after induction (see Fig. 2B). (b) Loss of plasmid following overnight growth (see Fig. 2B). (c) Ceils expressing proteinase became increasingly viscous and difficult to process with an extended growth period. (d) As the product was unstable losses would be minimised with a short cell period. Before further scale up the level of IPTG was optimised at the 4 1 scale, the final level being fixed at 1 mM. The optimised process at the 450 1 scale is illustrated by Fig. 3. Typically, at induction (6 h) the ODs50 was 3-7 with cells harvested some 3 h later to coincide with peak proteinase activity. No detectable activity was observed prior to induction. A marked decrease in CO 2 production (a measure of cells undergoing active respiration) coincided with the start of proteinase expression again highlighting the deleterious nature of the expressed product. It is possible that the proteinase uses a key cellular component as a substrate leading to a loss of cellular activity. Production at the 450 1 scale typically yielded 3-5 kg wet wt of cells.
132 .C
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~
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Fig. 3. Expression of HIVP at 450 1 scale. Conditions are defined in Fig. 1. HIVP expression (e) upon induction leads to toxicity as shown by a slowing down in growth ( • ) and a drop in exhaust CO 2 (o).
Purification A small-scale method for production of 50 p.g HIVP was developed before initiation of scale-up (Montgomery et al., 1991), comprising acetone precipitation of crude cell lysate (Hansen et aI., 1988), ion-exchange (IEC), hydrophobic interaction on alkyl Superose (HIC) and gel permeation chromatography (GPC). All of these steps proved to be scaleable from Pharmacia FPLC to BioPilot scale. Acetone precipitation was retained as a first step since it improved the recovery of enzyme, possibly due to release of HIVP loosely associated with lipid. Subsequent extraction of acetone powder (which can be stored for several months at - 7 0 °C without loss of activity) with 0.5 M NaCl/0.1% Triton X-100/40% glycerol pH 6.0 is a critical stage; inadequate homogenising of the acetone powder leads to HIVP remaining aggregated and voiding from the subsequent IEC column. Extracted acetone powder was diluted and immediately loaded onto the ion exchange column followed by reverse elution with an NaCl gradient (Fig. 4A). Pooled active fractions were then made up to 1.6 M ammonium sulphate at the head of the hydrophobic interaction column; 1.6 M ammonium sulphate is close to the salting-out point of HIVP but this concentration was found to be necessary for good retention on HIC. Fractions from the HIC column were eluted into glycerol to preserve activity, concentrated overnight 5-fold and loaded onto Superdex G75 gel filtration (GPC) column pre-equilibrated in 50 mM Hepes, pH 6.0, containing 0.5 M NaC1, 1 mM DTI', 2 mM EDTA (Fig. 4B); proteinase activity elutes at around 11 kDa molecular mass under these salt conditions, which may be due to dissociation to the monomer in the absence of substrate (Singh et al., 1990), although ultracentrifugation evidence suggests a stable dimeric form under similar conditions (Meek et al., 1989). The GPC stage affords a 2.7-fold purification. The overall purification
133 13.2 -
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0.2-
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~o
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Elution Vol (ml) 8.~8-
0.06-
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¢ 4,44-
0.04-
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0.02 •
B
128 K
44 K
17 K
135 K
:E
O-
0
600
800 1000 Elution Vol (ml)
1200
1400
1600
1800
Fig. 4. (A) Ion exchange chromatography of solubilised acetone powder on S-Sepharose Fast Flow, HIVP activity (o) is eluted by 0.5-1 M NaCI and loaded onto HIC after addition of (NH4)2SO 4. (B) Gel permeation chromatography of concentrated ex HIC pool on Superdex G75, the final step in purification. Active fractions (e) are 90-95% pure HIVP; inhibitors for co-crystallisation are added at this stage. TABLE 1 Purification of HIV-1 proteinase from 500 g wet weight cells
Disrupted cells Acetone powder Ex IEC Ex HIC * Ex GPC
Protein (mg ml - t)
Volume (ml)
Total protein (mg)
18.4
2200
40480
2.84
1800
5107
0.57 0.90 0.039
1420 152 782
812 113 30.7
Sp. Act, (nmoles min - 1) min mg - l )
Total Act. (tzmoles min- l)
Recovered Act. (%)
2243
100
470
2 400
107
2158 10408 27 737
1752 1176 744
78 52 33
55.4
* GPC load was concentrated to 100 ml on an Amicon YM10 and loaded in two 50 ml runs onto GPC.
134 M'W
K -
1
2
3
4
o
.o
•
K
5
Fig. 5. Silver-stained SDS-PAGE of purified HIVP. Tracks 2, 4; molecular weight markers. Tracks 1, 3, 5; 500 rig, 250 ng and 125 ng of HIVP respectively. The major band at 11 kDa is consistent with the expected molecular weight.
20
~ 15fe
-_= 8C 8 G)
_o= u_ 5-
o 300
~ 320
340 360 380 400 Emission wavelength (nm)
420
Fig. 6. Fluorescence spectrum of HIVP, excitation at 285 nm. The emission spectrum is dominated by the two tryptophans which are largely exposed, according to the crystal structure; the emission mmdmum (345 nm) is in agreement with this observation.
135
scheme starting with 500 g cells is summarised in Table 1; in order to get good overall recovery and yield it was essential to keep columns and buffers cold, use MilliQ water, keep glycerol/DTT in the solvent, work as quickly as possible (the whole process could be completed in 2.5 d) and finally, before concentration for structural studies, it is necessary to inhibit the enzyme to prevent autodegradation. The overall yield is, as far as can be ascertained, comparable to previous reports, also from E. coli (McKeever et al., 1989; Danley et al., 1989). Several hundred mg of the HIVP have been obtained by this process. The protein is 90-95% pure HIVP as judged by SDS-PAGE (Fig. 5). N-terminal sequence analysis by Edman degradation confirmed the correct N-terminus and high purity of the preparation. The specific activity is about 75% the value found for HIVP purified on the analytical scale (Montgomery et al., 1991), suggesting some inactivation has occurred during the longer preparative procedure in spite of the precautions taken. Fluorescence spectroscopy (Fig. 6) is another useful monitor of protein integrity; HIVP has two tryptophans, one of which, Trp6, is fully exposed and the other, Trp42, slightly buried according to the crystal structures (Wlodawer et al., 1989; Miller et al., 1989). The fluorescence spectrum is consistent with this structural data; Trp fluorescence decreases in emission wavelength in apolar environments (Campbell and Dwek, 1984). Maximal emission at 345 nm (Fig. 6) is a little below the value of 352 nm expected for fully exposed Trp. A further indication of the quality of the protein comes from its crystallisation. HIVP prepared as described was found to crystallise in 1 M NaCI pH 7.0 from hanging drops; crystals were tetragonal (P41) and diffracted to 2.7/~ at the SERC Daresbury Synchrotron source, and are thus essentially the same as crystals first prepared by McKeever et al. (1989). Co-crystals with proprietary ligands are now being used for diffraction studies.
Acknowledgements Many thanks to Mr. G.M. Turcatti, Glaxo Institute of Molecular Biology, Geneva for N-terminal sequencing, to Miss E. Roud-Mayne and Mr. J. Piercey for help in purification and to Dr. T.J.R. Harris, Dr. A.N. Hobden and Dr. M.J. Dawson for helpful comments.
References Campbell, I.D. and Dwek, R.A. (1984) Fluorescence, Chapter 5. In Biological Spectroscopy, Benjamin, Cummings Publishing Company Inc. Danley, D.E., Geoghegan, K.F., Scheld, K.G., Lee, S.E., Merson, J.R., Hawrylik, S.J., Rickett, G.A., Ammiratic, M.J. and Hobart, P.M. (1989) Crystallisable HIV-1 protease derived from expression of the viral pol gene in Escherichia coll. Biochem. Biophys. Res. Comm. 165, 1043-1050. Darke, P.L., Leu, C-T., Davis, L.J., Heinbach, J.C., Diehl, R.E., Hill, W.S., Dixon, R.A.F. and Sigal, I.S. (1989) HIV protease: bacterial expression and characterisation of the purified aspartic protease. J. Biol. Chem. 264, 2307-2312.
136 Erickson, J., Neidhart, D.J., Van Drie, J., Kempf, D.J., Wang, X.C., Norbeck, D.W., Plattner, J.J., Rittenhouse, J.W., Turon, M., Wideburg, N., Kohlbrenner, W.E., Simmer, R., Helfrich, R., Paul, D.A. and Knigge, M. (1990) Design, activity and 2.8 A crystal structure of C 2 symmetric inhibitor complexed to HIV-1 protease. Science 249, 527-533. Hansen, J., Billich, S., Schulze, T., Sukrow, S. and Moelling, K. (1988) Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody. Eur. Mol. Biol. Org. J. 7, 1785-1791. Kohl, N.E., Emini, E.A., Schlief, W.A., Davis, L.J., Heinbech, J.C., Dixon, R.A.F., Scolnick, E.M. and Sigal, I.S. (1988) Active HIV protease is required for viral infectivity. Proc. Natl. Acad. Sci. U.S.A. 85, 4686-4690. McKeever, B.M., Navia, M., Fitzgerald, P.M.D., Springer, J.P., Leu, C.T., Heinbach, J.C., Herber, W.K., Sigal, I.S. and Darke, P.L. (1989) Crystallisation of the aspartyl-protease from HIV-1. J. Biol. Chem. 264, 1919-1921. McQuade, T.J., Tomasselli, A.G., Liu, L., Karacostas, V., Moss, B., Sawyer, T.K., Henrikson, R.L. and Tarpley, W.G. (1990) A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation. Science 247, 454-456. Meek, T.D., Dayton, B.D., Metcalf, B.W., Dreyer, G.B., Strickler, J.E., Gorniak, J.G., Rosenberg, M., Moore, M.L., Magaard, V.W. and Debouck, C. (1989) HIV-1 protease expressed in E. coli behaves as a dimeric aspastic protease. Proc. Natl. Acad. Sci. U.S.A. 86, 1841-1845. Miller, M., Schneider, J., Sathyanarayanara, B.K., Toth, M.V., Marshall, G.R., Clawson, L., Selk, L., Kent, S.B.H. and Wlodawer, A. (1989) Structure of complex of synthetic HIV protease with a substrate-based inhibitor at 2.3 A resolution. Science 246, 1149-1152. Montgomery, D.S., Singh, O.M.P., Gray, N.M., Dykes, C.W., Weir, M.P. and Hobden, A.N. (1991) Expression of an autoprocessing Cat-HIV1 proteinase fusion protein: purification to homogeneity of the released 99 residue proteinase. Biochem. Biophys. Res. Comm., submitted for publication. Pearl, L.H. and Taylor, W.R. (1987) A structural model for the retroviral proteases. Nature 329, 351-354. Singh, O.M.P., Roud-Mayne, E. and Weir, M.P. (1990) Dimerisation of HIV-1 protease, In: Pearl, L.H., Ed., Retroviral proteases, Maturation and Morphogenesis, Macmillan Press Ltd., N.Y., U.S.A., pp. 73-78. Strickler, J.E., Gorniak, J., Dayton, B., Meek, T., Moore, M., Magaard, V., Malinowski, J. and Debouck, C. (1989) Characterisation and autoprocessing of mature forms of HIV-1 protease from E. coli. Proteins 6, 139-154. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B.K., Baldwin, E., Weber, I.T., Selk, L.M., Clawson, L., Schneider, J. and Kent, S.B.H. (1989) Conserved folding in retroviral proteases: crystal structure of synthetic HIV-1 protease. Science 245, 616-621.
