PROTEIN

EXPRESSION

AND

3, 313-321

PURIFICATION

(19%)

Production, Purification, and Characterization of Human Recombinant IL-8 from the Eucaryotic Vector Expression System Baculovirus Xiao-Qiang

Kang,

Jonkeeta

Department of Biochemistry of North Texas, Fort Worth,

Received

October

Wiggins,

Sushanta

Mallick,

and Stephen

and Molecular Biology, Texas College of Osteopathic Texas 76107-2960 ~-

R. Granti

MedicinelUniversity

30,199l

The cDNA for the human chemotactic interleukin, IL-8 was subcloned from a bacterial source into the eucaryotic vector expression system baculovirus. Recombinant human IL-8 (rhIL-8) was synthesized and secreted from Sf9 cells derived from Spodoptera frugiperda following infection of a recombinant virus harboring the full-length IL-8 structural gene. Infected Sf9 cells produced rhIL-8 in a range from 0.5 to 2.0 mg of rhIL4Vliter of postinfection cell culture media. The recombinant interleukin was purified (>600-fold) to homogeneity using preparative HPLC. rhIL-8 retained all of the physical, immunological, and biochemical properties observed for the natural product, monocyte-derived IL-8. rhIL-8 was assessed for biological efficacy by three criteria: (a) ability to induce chemotaxis in human neutrophils, (b) ability to induce oxygen burst metabolism, and (c) ability to be recognized by purified rabbit antibody generated against monocyte-derived IL-8. Antibody generated against monocyte-derived IL8 recognized rhIL-8 isolated during all stages of the purification protocol. rhIL-8 was strongly chemotactic for human neutrophils and exhibited a chemotactic index comparable to that reported for other strong chemotactic peptides. rhIL-8 was identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single silver-stained band having an estimated molecular weight of 9.2 kDa and displayed amino acid residue molar abundance homology predicted for the mature form of the interleukin. Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production. 0 1992 Academic Press, Inc.

’ To whom correspondence Biochemistry and Molecular Medicine, 3500 Camp Bowie (817) 735-2133.

should be addressed at Department of Biology, Texas College of Osteopathic Blvd., Fort Worth, TX 76107-2960. Fax:

1046-5928192 $5.00 Copyright D 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Chemotaxis, directed cell migration in response to a chemical signal, plays an important role in the primary immune system in humans. A variety of chemical signals that are shared by phagocytic cells of the immune system have been identified. Recently, the first human interleukin demonstrating chemotactic activity for neutrophils was identified (l-3) and named MDNCF2 for monocyte-derived neutrophil chemotactic factor. MDNCF was renamed IL-8 because it was synthesized in several cell types and was shown to contain chemotactic properties for more than one cell type (45). IL-8 has been purified to homogeneity (6), its cDNA cloned into bacteria, and the recombinant bacterial product characterized (7). More recently, a genomic clone has been isolated (8--g), including characterization of the 5’ upstream regulatory region (10). IL-8 has little sequence homology to any of the identified human interleukins (11). However, the 5’ flanking region contains several recognized regulatory &-acting sites shared between three unique groups of cytokine gene families: acute-phase response genes, genes of inflammatory response, and pluripotential activating interleukins (1214). Studies over the past 2 years using bacterial recombinant IL-8 have resulted in the partial characterization of the IL-8 receptor (15-21). We are interested in human neutrophil chemotaxis signaling and want to explore cellular mechanism(s) which control the level of IL-8 receptor expression dur-

’ Abbreviations used: rhIL-8, recombinant human interleukin 8; Sf9, Spodopterafragiperda; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; MDNCF, monocyte-derived neutrophi1 chemotactic factor; HPLC, high-performance liquid chromatography; Mes, 2-(N-morpholino)ethanesulfonic acid, AcNPV, Autographa californica nuclear polyhedrosis virus; CIAP, calf intestine alkaline phosphatase; ELISA, enzyme-linked immunosorbent assay; FLMP, N-formyl-Leu-Met-Phe; TFA, trifluoroacetic acid; TPCK, L-l-p-tosylamino-2-phenylethylchloromethyl ketone. 313

314

KANG

ing human neutrophil maturation. This interest has led us to develop a readily available source for large amounts of chemotactically active human IL-8. Expression of human cDNA in a bacterial expression system does not result in a mature form of the recombinant gene product. Signal sequences are not removed and most post-translational processing observed in mammalian systems does not take place in bacteria. Often this incomplete processing of nascent protein leads to poor biological activity. Therefore, we have subcloned the entire structural portion of the human IL-8 gene from a bacterial gene construct into a shuttle vector specific for the eucaryotic vector expression system baculovirus. The overall goals of this project are: (i) to produce large amounts (>l.O mg of interleukin/liter of conditioned media) of recombinant human IL-B, (ii) to obtain chemotactically viable recombinant human IL-8 whose biological properties match those of the monocyte-derived natural product, and (iii) to develop a purification protocol for rhIL-8 involving preparative HPLC technology. This report describes the synthesis and secretion of recombinant human IL-8 from the baculovirus vector expression system, purification by preparative HPLC, and immunological and biological characterization of this important chemotactic interleukin. MATERIALS

Cells and viral stocks. Spodoptera frugiperda (Sf9) armyworm insect cells, Autographa californica nuclear polyhedrosis virus (AcNPV), and pVL1392-3 were gifts from the laboratory of Dr. Max Summers (Texas A&M University, College Station, TX) (22). The bacterial vector construct, (yMDNCF2-l)pUCl9-1.7-5, harboring the full-length structural portion of the human IL-8 gene (7), was a gift from Dr. Kouji Matsushima at the National Cancer Institute. Escherichia coli DH5a (sup E44, hsd R17, recA1, endAl,gyrA96, thi-1, relA1) cells were obtained from GIBCO/BRL. Insect Sf9 cells were cultured in chemically defined serum-free GIBCO media, Sf900 containing 50 pg of gentamycin sulfate and 2.5 pg amphotericin B (Fungizone) per liter. Cells were grown in T flasks (175 cc) containing 30 ml of Sf900 media and maintained at 28°C in a sterile air atmosphere. Viral infections were made using 30 flasks (175 cm2) of Sf9 cells at 70% confluence. Each flask (2.0 X lo7 cells) was infected with 1 ml of recombinant virus stock (>105 pfu/ml) diluted into 10 ml of culture media. Chromatographic materials. Chromatography was performed on a Waters 600E preparative HPLC equipped with a variable-wavelength detector and controlled by computer-interfaced Baseline Powerline interfacing software. Preparative HPLC columns used were TSK-SP-5PW (SP cation-exchange resin), 21.5 X 150 mm; Protein-Pak 125, 10 X 30 mm (three columns

ET AL.

in series); and Protein-Pak C4,5 pm (reverse-phase matrix), 7.8 x 150 mm (Waters, Inc.). SDS-PAGE was carried out using ultrapure acrylamide and bisacrylamide from GIBCO/BRL. Other materials. Restriction endonucleases were purchased from GIBCO/BRL and used as recommended by the supplier. All reagents for amino acid composition analysis were obtained from Pierce Inc. Fluorescent substrates for oxygen burst metabolism assay were from Molecular Probes. Chromatographic solvents were from Burdick and Jackson Laboratories. All other reagents were of A.C.S. grade or better and supplied by Sigma. METHODS

Subcloning human IL-8 into the baculovirus shuttle vector, pVL1392. The structural portion of the human IL-8 gene was restriction endonuclease excised from a bacterial vector construct following plasmid transfer (transfection) and gene amplification in E. coli DH5a cells. The subcloning strategy, schematically illustrated in Fig. 1, involved BamHI digestion of (TMDNCFBl)pUC19-1.7-5 to yield a 900-bp restriction fragment. cDNA fragments were separated on a 0.7% ultrapure low-temperature melting agarose gel by electrophoresis in Tris/borate/EDTA buffer as described by Sambrook et al. (23). The agarose slice containing the cDNA insert was melted and contaminating proteins were removed by conventional organic extraction followed by ethanol precipitation as described by Sambrook et al. (23). Following BamHI and CIAP digestion, the cleaved shuttle plasmid, pVL1392, was electrophoretically isolated on a low-melting agarose gel and contaminating proteins were removed as described above. Ligation was performed using 0.5 pg of insert and 5.0 pugof shuttle plasmid cDNA by the protocol of King and Blakesley (24). An aliquot of the ligation mix was electrophoretically analyzed to ensure complete ligation. E. coli DH5a cells were transformed with the shuttle plasmid construct and the harbored interleukin gene was amplified in the transformants in the presence of the selectable marker, ampicillin (75 pg/liter of LB broth). cDNA IL-8 insert was integrated into the polylinker region of the baculovirus shuttle vector pVL1392 at a unique BamHI site located a few bases downstream from the start site for RNA synthesis and positioned immediately behind the strong baculoviral polyhedron promoter. Subcloned transformants were evaluated for correct gene orientation by asymmetric cutting with appropriate restriction endonucleases and frozen as 20% glycerol stocks. A single transformants was grown to a 2-liter culture and the shuttle plasmid construct containing IL-8 was purified using the mild alkaline hydrolysis method for cDNA purification as described by Sambrook et al. (23). The purified construct was again evaluated for correct insert ori-

HUMAN

1

1Kb

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AND

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315

BACULOVIRUS

BaculovlrusShuttle Vector

PUC141.7~5

Pat1

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IL-8

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Human IL-9

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(pVL1392-11-8) pVL1392

I FIG. 1. baculovirus

Schematic shuttle

showing the strategy plasmid pLV1392.

for subcloning

Human IL-8

the structural

entation, plasmid purity, and cDNA mass and frozen at -20°C as 20% glycerol stocks. These -2O’C stocks were routinely used to produce recombinant virus. Recombinant baculovirus production. The production and purification of recombinant baculovirus stock harboring the human IL-8 cDNA was performed according to the protocol developed by Invitrogen Corp. (San Diego, CA). Following amplification in E. coli, the purified shuttle vector-human IL-8 construct was incubated for 2 h at 28°C with wild-type baculovirus in subconfluent cultures of the armyworm insect cell line, Sf9. After removal of excess virus and DNA, fresh medium was added to a final volume of 5 ml per 60-mm well in six-well plates. The cultures were incubated at 28°C for 7-10 days and the cell supernatant solution was harvested by centrifugation at 15OOg for 20 min to remove lysed cell debris. The mixed viral stock solution was stored aseptically at 4°C awaiting further viral subcloning. Serial dilutions (10-l to 10-s) of the mixed viral stock were incubated with subconfluent cultures of Sf9 cells for 2 h. The excess virus was removed and individual cultures of virus-infected Sf9 cells were overlaid with 1.25% low-melting agarose. Five to seven days later the cultures were stained with neutral red dye and recombinant plaques were selected on an inverted micro-

I I

portion

of human

IL-8

cDNA

from

a bacterial

plasmid

source

to the

scope. Selected recombinant plaques were amplified and subcloned as described above. This process was repeated two additional times. The procedure yielded monoclonal recombinant viral stocks free of any contaminating wild-type plaques. Positive clones were evaluated using a conventional ELISA assay described below 8-11 days postinfection. Chromatographic methods. All buffers were prefiltered through 0.22~pm Millex filters (Millipore, NY) and degassed prior to use. All chromatography was performed under an argon purging atmosphere and protein was monitored at 280 nm for all columns except for the reverse-phase Delta-Pak C4 column, which was monitored at 218 nm. The protocol for the purification of recombinant human IL-8 synthesized in the baculovirus vector expression system is outlined in Fig. 2. Chromatographic elution conditions for each of the preparative HPLC columns were as described in the figure legends. Anti-IL-8 ELISA using antibody generated against monocyte-derived IL-8. Aliquots (100 ~1) of even-numbered column elution fractions were delivered to flatbottomed 96-well Falcon tissue culture plates and assayed for rhIL-8 using a conventional ELISA (25) capable of detecting 0.1-0.4 ng of the recombinant interleukin per well. Following antigen binding and

316 Conditioned

KANG media (1 liter) from recombinant

virus(hlL-8)

infected

sf9 cells

t CM-Catton Exchange Chromatography on ToyoPearl TSK-CM-650 (fast flow)

SP-Cation Exchange Chromatography on TSK-SPSPW HPLC Column

t C-4 Reverse Phase Chromatography on Delta-Pak RP-C-4 (3WA) Column

1 Gel Filtration HPLC Chromatography on (3x) Protein-Pak 125 Column

t C4 Reverse Phase Chromatography on Detta Pak RP-C4 (-A) Column Schematic showing recombinant human IL-8.

an outline

their ability to induce human neutrophil chemotaxis by the method of Nelson and Herron (29). The chemotactic index is defined as index

= chemotactic

I

for

AL.

chemotactic

1

FIG. 2.

ET

of the purification

protocol

blocking of the nonspecific sites with diluted milk, plates were washed and incubated with rabbit anti-human IL-8 antibody at a 1500 dilution for 2 h. The excess antibody was washed and the primary immunoconjugate was incubated for 2 h with a peroxidase-conjugated second antibody, goat anti-rabbit IgG at a 1:500 dilution. Following removal of the second conjugated antibody and final wash, the ELISA color was developed by adding 100 ~1 of a freshly prepared 1 mg/ml solution of 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) in 10 mM sodium acetate, pH 5.0, containing 40 ~1 of 3.0% hydrogen peroxide. Samples were analyzed using a Bio-Rad ELISA plate reader at 415 nm. Positive (purified commercial IL-8 from monocytes) and negative (nonspecific antigen, no antigen, no first and/or second antibody added) controls and all fraction eluates were assayed in triplicate. Denaturing SDS-PAGE. Even-numbered column fractions were subjected to gradient gel electrophoresis. SDS-PAGE was performed in the presence of p-mercaptoethanol by a modification of the method of Laemmli (26). The modification consisted of lowering the bisacrylamide:acrylamide ratio from the standard l-0%:28% (w/w) to 0.5%:40% (w/w). Samples subjected to SDS-PAGE were visualized by silver staining using the method of Merill et al. (27). Human neutrophil isolation. Human neutrophils were isolated from donor blood (50 ml) of healthy adult male as described by the method of Dooley et al. (28). Human neutrophil chemotaxis assay. Column fractions showing activity by ELISA were evaluated for

distance - random motility distance random distance motility Superoxide anion production assay. The method of Allen et al. (30) was employed to assess superoxide anion nroduction bv human neutronhils stimulated either by FMLP, a synthetic chemotactic peptide (positive control), or purified rhIL-8, or column fractions containing rhIL-8. Oxygen burst metabolism assay. Oxygen burst metabolism was determined using dihydrodichlorofluorescein as a sensitive fluorogenic chromophore to follow the oxidation of the nonfluorescent substrate, dihydrodiacetylflouroescein, to its fluorescent product, dihydrofluorescein as developed by Wymann et al. (31). This assay consumes hydrogen peroxide secreted by human neutrophils exposed to the chemotactic agent and requires saturating amounts of exogenous horseradish peroxidase to be present during the oxidation of the substrate. The assay detects oxygen burst metabolism in the picomole range of sensitivity. Amino acid analysis. Protein samples were subjected to denaturing SDS-PAGE and electrophoretitally transferred to Immobilon P membrane (Millipore Corp., NJ) by the method of LeGendre and Matsudaira (32) using a Bio-Rad electrophoretic transfer apparatus. The transferred proteins were stained for 2 min with Coomassie brilliant blue in 50% methanol and destained in 50% methanol, and the stained band was carefully cut away from the Immobilon P support. Samples were hydrolyzed in vacua at 1lO’C for 48 and 72 h in 6 N HCl. Hydrolyzed samples were assayed for amino acid composition by the method of Dong and Gant (33). Values were normalized for total molar abundance and compared to values for the mature form of human IL-8 based on cDNA sequence (7). RESULTS

Subcloning of human IL-8 and recombinant virus production. A strategy for the subcloning of the structural portion of human IL-8 cDNA from a bacterial plasmid source to the baculovirus shuttle plasmid pVL1392 is shown schematically in Fig. 1. Transformants were selected and subcloned, and plasmid DNA was carefully analyzed by agarose gel electrophoresis following restriction endonuclease digestion on selected unique restriction sites within the structural portion of the insert and its flanking polylinker region. Recombinant viral plaques were purified to monoclonality through three plaque isolation procedures as described under Mate-

HUMAN

IL-8

AND

BACULOVIRUS

f I

0.00 Fraction

No. ( 4 ml&.

)

0.50

1.00

I

1.50

x 102 minutes

FIG. IL-8

3. Cation-exchange chromatography of lo-day postinfection conditioned media following recombinant virus infection of the human gene. (A) Chromatography (cation-exchange fast-flow) on ToyoPearl TSK-CM 650 column (75 X 250 mm) was as follows: Buffer A, 20 mM Mes, pH 6.0; Buffer B, 20 mM Mes, 500 mM NaCl, pH 6.0. Isocratic elution was in 100% Buffer A for 500 ml, linear gradient to 100% B, 1000 ml, followed by isocratic elution in 100% Buffer B for 300 ml. The sample (200 ml) harvested from conditioned media (7 days postinfection) was dialyzed, concentrated, and loaded onto the ToyoPearl TSK-CM 650 column (800 ml total bed volume). The fractions (8 ml) were collected following monitoring (O.D. at 280 nm). (B) Chromatography on the TSK-SP-5PW column was as follows: Buffer A, 20 mM Mes, pH 6.0; Buffer B, 20 mM Mes, 500 mM NaCl, pH 6.0. The active fractions from the CM column were concentrated (40 ml) and dialyzed against Buffer A. The sample was loaded onto the TSK-SP-5PW column pre-equilibrated with Buffer A with two successive 20-ml injections. Elution conditions were 2.0 ml/min flow rate using the following gradient conditions: isocratic elution in 100% Buffer A for t = O-20 min; linear gradient to 100% B, t = 20-180 min; isocratic in 100% Buffer B, t = 180-210 min. The eluate was monitored at 280 nm and fractions, 2 min (4 ml), were collected. The results of an ELISA on all even-numbered fractions has been included in the elution profile of the SP column.

rials and Methods. Recombinant virus harboring human IL-8 cDNA was amplified in Sf9 and harvested late in the near-lytic stage of infection. Harvested recombinant viral stocks were used to infect large-scale cultures of Sf9 cells for production of recombinant human IL-8. Purification of recombinant human IL-8. The purification protocol employing preparative HPLC technology is outlined in Fig. 2. Analysis of a typical l-liter culture for the purification of recombinant IL-8 from the recombinant virus-infected Sf9 cells is presented below. Conditioned medium from an infected culture containing 450 mg of total protein was harvested 8-10 days postinfection by centrifugation at 1500g for 10 min and loaded onto a ToyoPearl TSK-CM 650 column. Shown in Fig. 3A is the elution profile of the cell supernatant from the fast-flow carboxymethyl ion-exchange column. A majority of the protein did not bind to the column and eluted early in the void volume fractions. A UV-absorbing protein peak containing rhIL-8 eluted late in the gradient elution profile at 400 mM NaCl(lOO0 mho conductivity). This fraction contained a large amount (> 1.0 mg) of rhIL-8 as evaluated by ELISA and silver staining. Biological activity was determined using

freshly isolated human neutrophils in the agarose chemotaxis assay as described under Materials and Methods. The CM column removed 80% of contaminating proteins. ELISA-positive fractions (160-180) were pooled, concentrated, dialyzed against Buffer A of the SP column, and loaded onto a TSK-SP-5PW column. As shown in Fig. 3B, rhIL-8 eluted late in the linear gradient portion of the column profile (fractions 113 to 119). rhIL-8 was once again identified by ELISA and silver staining following SDS-PAGE. Results from ELISA have also been included in Fig. 3B. Fractions 113 to 119 were pooled, dialyzed against Buffer A of a reverse-phase Delta-Pak RP-C4 (300A) column, and loaded. Shown in Fig. 4A is the elution profile of rhIL-8. Importantly, this column resolved more than 10 protein contaminants which coeluted with IL-8 on the previous SP column. Fractions 34-39 contained a large amount (>0.5 mg) of antibody-reactive rhIL-8. The pooled fractions were dialyzed against a buffer used for HPLC 3X Protein-Pak 125-PW gel filtration column, concentrated to a volume of 1 ml, and then loaded onto the column. As shown in Fig. 4B, a major UV-absorbing peak containing antibody-reactive rhIL-8 was eluted be-

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IL-8

1

0.00

2.00

4.00 x 10’ minutes

6.00

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FIG. 4. Reverse-phase and gel filtration HPLC chromatography of recombinant human IL&containing fractions. (A) Chromatography on the reverse-phase Delta-Pak RP-C4 (300A) column (4.9 X 150 mm) was as follows: Buffer A, 0.1% TFA in H,O; Buffer B, 60% acetyl nitrile in H,O containing 0.1% TFA. Elution conditions were 0.5 ml/min flow rate collecting 0.5-ml fractions using the following gradient conditions: isocratic elution in 100% Buffer A for t = O-20 min; linear gradient to 100% Buffer B, t = 20-80 min; isocratic in 100% Buffer B, t = 80-90. (B) Chromatography on Protein-Pak 125-PW column was as follows: elution buffer, 20 mM Mes, pH 6.0, 100 mM NaCl, pH 6.0, containing 100 pg ovalbumin. Elution conditions were 0.5 ml/min flow rate in elution buffer for t = O-90 min.

tween fractions 39 and 45. The majority of protein in these fractions proved to be rhIL-8 as judged by ELISA and silver staining following SDS-PAGE with an esti-

I- 1.0

T

mated molecular weight of 8-12 kDa. Fractions 39-45 were pooled, concentrated, and dialyzed against loading buffer of a second reverse-phase column. The sample was loaded onto the Delta-Pak RP-C4 (300A) column and a shallow linear gradient was generated. Three UVabsorbing peaks eluted in the linear portion of the gradient as shown in Fig. 5. rhIL-8 eluted as a single sym-

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FIG. 5. Chromatography on reverse-phase Delta-Pak RP-C4 (300A) column (4.9 X 150 mm) using a shallow gradient. Chromatography was as follows: Buffer A, 10% acetyl nitrile containing 0.1% TFA in H,O, Buffer B, 60% acetyl nitrile in H,O containing 0.1% TFA. Elution conditions were 0.5 ml/min flow rate using the following gradient conditions: isocratic elution in 100% Buffer A for t = O-20 min; linear gradient to 70% Buffer B, t = 20-80 min; isocratic in 70% Buffer B, t = SO-90 min. The results of an ELISA on even-numbered fractions have been included in the elution profile.

FIG. 6. Silver-stained protein following denaturing SDS-PAGE in the presence of fl-mercaptoethanol. A gradient gel of lo-20% acrylamide was performed as described under Materials and Methods: lane 1, aliquot (50 ~1) of pooled fractions 57 and 58 from the shallow gradient reverse-phase column profile shown in Fig. 5; lane 2, blank, lane 3, molecular weight standards.

HUMAN TABLE

Purification

AND

1

of Human Recombinant IL-8 by HPLC Volume (ml)

Procedure Cell supernatant Post-ToyoPearl Post-SP-TSK-5PW Post-Delta-Pak

IL-8

CM C4-RP

1000 200 10 4

Total

protein bd 367.3 39.6 3.2 0.6

Purification (fold)

319

BACULOVIRUS

cyte-derived human IL-8 at all stages of purification. Purified rhIL-8 showed high chemotactic response in the agarose chemotaxis assay. rhIL-8 had a chemotactic index similar to that of other strong neutrophil active chemotactic agents such as FLMP.

9.3 115.2 628.9

metrical peak. As shown in Fig. 6, fractions 57-58 contained pure rhIL-8 as visualized by silver staining following SDS-PAGE. It is clearly evident that the pooled fractions 57 and 58 contain a single protein with an estimated molecular weight of 8.7 kDa. rhIL-8 and other resolved proteins from the RP-C4 column were individually pooled and critically evaluated for their ability to (a) stimulate chemotaxis of human neutrophils, (b) induce oxygen burst metabolism in human neutrophils, and (c) react with polyclonal antibody generated against IL-8 purified from human monocytes. Biochemical and immunological characterization of rhIL-8. A summary of the results of the purification of rhIL-8, outlined in Fig. 2, is shown in Table 1. The recombinant interleukin was purified over 600-fold with a yield in excess of 500 pg of rhIL-8 from 1 liter of infected culture. Purified rhIL-8 was subjected to amino acid compositional analysis. The results of that analysis and a comparison with the predicted composition of the mature form of monocyte-derived human IL-8 are presented in Table 2. Amino acid compositional analysis demonstrated that 14 out of 15 amino acids analyzed showed an amino acid residue abundance within 2 or less residue equivalence for that predicted from the cDNA sequence. Eight amino acids analyzed proved to have a residue abundance within a single molar equivalence to that expected. Finally, 5 out of 15 amino acid residues demonstrated an amino acid residue composition identical to that predicted from cDNA. Several attempts were made to microsequence purified rhIL-8. Unfortunately, sequencing results suggested that rhIL8 may be N-terminally blocked. Therefore, rhIL-8 was exhaustively digested with TPCK-trypsin and peptides were isolated using RP-Cl8 chromatography. The digestion yielded a peptide elution profile identical to that predicted for TPCK-trypsin-digested IL-8 based upon cDNA sequence information (data not shown). Microsequencing of a single peptide eluting from the Cl8 column correctly identified 4 of the first 7 amino acids in the peptide sequence. Results of the immunological characterization of purified rhIL-8 and a comparison to other chemotactic agents are shown in Table 3. The recombinant interleukin was consistently immunoreactive against rabbit antibody generated against mono-

DISCUSSION The goals of this project, to produce large amounts (>l.O mg of interleukin/liter of conditioned media) of recombinant human IL-8, to obtain chemotactically viable recombinant human IL-8, and to develop a purification protocol for rhIL-8 involving preparative HPLC technology, have all been successfully achieved. The question of whether the baculovirus vector expression system is capable of producing a biologically viable (chemotaxis-active) human IL-8 in large amounts has been addressed in Tables 1 and 3. It is evident that rhIL-8, synthesized and secreted from postinfection Sf 9 cells, was produced in amounts exceeding 500 pg/liter of postinfection conditioned media. This recombinant interleukin retains all of its biological properties and chemotactic activity. When used in picomolar concentrations, purified rhIL-8 displayed a chemotactic index in the range obtained for strong chemotactive peptides such as FLMP. Recombinant human IL-8 was purified greater than 600-fold from SfS-conditioned media. Results shown in Figs. 3, 4, and 5 confirm the usefulness and speed of preparative HPLC technology in the purification of rhIL-8. Results from Fig. 6 demonstrate that rhIL-8 was purified to homogeneity as judged by silver staining following denaturing SDS-PAGE. Summarizing the results in Table 2 combined with the results in Table 3 leaves little doubt that the silver-stained protein observed in Fig. 6 has an amino acid composi-

TABLE

2

Amino Acid Composition of Recombinant IL-8 Amino acid

IL-8 (nmol)

IL-8 residues

Expected

Asx Thr Ser Glx GUY Ala Val Met Ile Leu Tyr Phe His LYS Arg

0.250 0.162 0.325 0.456 0.000 0.150 0.139 0.052 0.128 0.193 0.127 0.082 0.091 0.213 0.187

6.0 3.9 7.8 10.9 0.0 3.6 3.3 1.3 3.1 4.6 3.0 2.0 2.2 5.1 4.5

5 2 5 10 0 3 5 0 5 6 1 3 2 9 5

IL-8 (W) 9.8 6.3 12.7 17.8 0.0 5.9 5.5 2.1 5.0 7.6 5.0 3.2 3.6 8.3 7.3

Expected (%I 8.2 3.3 8.2 16.4 0.0 4.9 8.2 0.0 a.2 9.8 1.6 4.9 3.3 14.8 8.2

320

KANG TABLE Biological

Properties

of Purified

ET

AL. 3 Human

Recombinant

IL-8 Oxygen

Chemotactant rhIL-8 IL-8/NAP-1 (monocyte) FMLP peptide

(2) 9200 9000 354

Antibody reactivity (anti-human IL-8 monocyte derived)” +++++ +++++ -/+

Chemotactic

index

1.67 1.87 2.17

Cytochrome assayb