Expression of recombinant feline tumor necrosis factor is toxic to ...

CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Nov. 1995, p. 740–746 1071-412X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 2, No. 6

Expression of Recombinant Feline Tumor Necrosis Factor Is Toxic to Escherichia coli CYNTHIA M. OTTO,1,2* FRANK NIAGRO,3 XINZHUAN SU,2

AND

CLARENCE A. RAWLINGS1,2

College of Veterinary Medicine, Department of Small Animal Medicine,1 Department of Veterinary Physiology and Pharmacology,2 and Department of Medical Microbiology,3 University of Georgia, Athens, Georgia Received 6 February 1995/Returned for modification 22 March 1995/Accepted 14 August 1995

The tumor necrosis factor (TNF) genes from cats, horses, and pigs have all been cloned into the pFLAG-1 fusion protein expression vector (International Biotechnologies, Inc., Kodak, New Haven, Conn.). Growth curves for Escherichia coli containing the pFLAG-1 vector alone and the pFLAG-1 vector containing the TNF gene from each species were determined by visible light spectrophotometry (at 600 nm). Porcine TNF, equine TNF, and feline TNF cultures had slower doubling rates than cultures containing the pFLAG-1 vector alone. Cultures of cells transformed with feline TNF reached peak densities at 3 to 4 h and then decreased to near initial densities prior to the recovery of growth. The induction of expression with isopropyl-b-D-thiogalactopyranoside (IPTG) arrested the growth of fresh feline TNF cultures for 6 h, which was followed by complete recovery. This inhibition occurred in two strains of E. coli (LL308 and JM101). Induced feline TNF cultures expressed the TNF-FLAG fusion protein for the first 6.5 h. Uninduced cultures expressed low levels of fusion protein. The feline TNF–pFLAG-1 vector was purified from cells expressing fusion protein and from cells with recovered growth curves. Sequencing the vector demonstrated the complete feline TNF gene and tac promoter in cells expressing the fusion protein and a deletional mutation of the tac promoter site in recovered cells. In contrast to equine and porcine TNF, the expression of recombinant feline TNF is toxic to E. coli. Alterations in protein folding and the prevention of secretion of the feline protein may explain the toxic effect.

critical evaluation of the effects of FTNF on the growth of E. coli. The purpose of this study was to determine the reason for the failure of the expression of an active FTNF protein in E. coli.

Tumor necrosis factor (TNF), a pluripotent cytokine, is produced primarily by macrophages (2, 8). It functions as an immune modulator under physiologic conditions (4, 11). In sepsis, ischemia reperfusion, and certain disease states, the acute overproduction of TNF contributes to multiple-organ failure and death (30, 34, 35). Chronic parasitemia, neoplasia, and some infections result in chronic TNF release and wasting syndromes (13, 15, 28). TNF has direct and indirect cytotoxic effects. It is capable of inducing cell-mediated bactericidal activity and cytotoxicity (3, 7). Direct cytotoxicity is seen with certain tumor cells, such as the WEHI 164 mouse fibrosarcoma clone (6), parasites (Pneumocystis carinii) (24), and viruses (11). There are no reports of direct toxic effects on bacteria. The TNF gene from multiple species has been expressed in Escherichia coli. The porcine and equine TNF genes have been successfully expressed with the pFLAG-1 fusion protein expression vector (International Biotechnologies, Inc. [IBI], Kodak, New Haven, Conn.) (31, 32). Multiple attempts to express the gene for feline TNF (FTNF) in the same system failed to consistently produce an identifiable fusion protein, and when a protein was obtained, it lacked bioactivity. Cells (both JM101 and LL308) transfected with (spliced genomic) FTNF that were plated and allowed to grow for 20 to 24 h resulted in ‘‘ghost’’ colonies. Colonies initially grew and then died, leaving a faint ring where the colony had been. Liquid cultures for minipreps obtained from the FTNF-transfected cells failed to grow within the standard 8-h culture time. The incubation of these cultures for 24 h demonstrated marked turbidity and bacterial growth. These findings led to a

MATERIALS AND METHODS Bacteria. Bacteria used were the E. coli strains DH5a, for propagation of the pFLAG-1 vector and for mutagenesis prior to expression, and JM101 and LL308 (IBI, Kodak), for protein expression. Culture conditions. Bacteria were grown at 378C in Luria-Bertani (LB) liquid medium or on LB plates. Cultures for the selection of bacteria containing the pFLAG-1 vector were grown in the presence of ampicillin (100 mg of ampicillin per ml of LB medium). Expression was initially conducted at 37, 30, and 228C. There was no difference in cell viability at these three temperatures, so all subsequent experiments were performed at 378C. The pFLAG-1 fusion protein expression vector was used. The pFLAG-1 vector contains a strong tac promoter, which is repressed by the vector lacI gene product and derepressed by isopropyl-b-D-thiogalactopyranoside (IPTG). Plasmid preparation, expression, and sequencing. The genomic FTNF DNA (20) was prepared for expression by a PCR splicing method as described by Su and coworkers for the expression of the porcine TNF gene (32). The sequences of the insert and the reading frame were confirmed by dideoxy sequencing. The pFLAG-1 vector (i) without the insert, (ii) containing the equine TNF gene, or (iii) containing the porcine TNF gene was also used to transfect LL308 and JM101 cells. The transfection of the competent (26) E. coli with the FLAG constructs was performed according to the methods of Sambrook et al. (26). Using agarose gel electrophoresis, we screened the plasmid DNA obtained by the rapid miniprep procedure as described by Zhou and others (36). Large quantities of the vector were obtained for sequencing by the recommended maxiprep procedure for the pZ 523 column (5 Prime-3 Prime, Paoli, Pa.). Isolation of the pFLAG-1 vector containing the FTNF gene was performed with uninduced E. coli and with E. coli that had shown growth inhibition and recovery. This double-stranded vector was sequenced by the dideoxy method with Sequenase enzyme (United States Biochemical, Cleveland, Ohio). Sequencing primers used were CATSEQ3 (an antisense primer synthesized by R. McGraw that is based on a known DNA sequence located in exon IV of FTNF) and those provided by IBI. The 59 IBI sequencing primer binding site was located at the tac promoter site. The 39 IBI primer binding site was located 39 of the multiple cloning site. Growth curves. Individual colonies containing the pFLAG-1 vector (without

* Corresponding author. Present address: School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-6010. Phone: (215) 898-3390. Fax: (215) 573-3925. Electronic mail address: [email protected]. 740

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FIG. 1. Effects of TNF expression on bacterial cell growth. The cell densities (OD600) of E. coli LL308 cells transfected with the pFLAG-1 vector are plotted over time. (A) Growth of cells transfected with the empty vector compared with growth of those containing the FTNF gene; (B) growth of cells transfected with the empty vector compared with growth of those containing the equine TNF gene; (C) growth of cells transfected with the empty vector compared with growth of those containing the porcine TNF gene. IPTG (0.75 mM) was added at 30 min to cultures marked with a plus sign for the induction of protein expression. All samples were assayed in triplicate.

inserted DNA) and the pFLAG-1 vector containing spliced genomic TNF (equine, feline, or porcine) were isolated and grown overnight in 2 ml of LB medium with ampicillin. Aliquots were taken from these overnight cultures and added to fresh LB medium with ampicillin to monitor growth curves. All cultures were grown in triplicate. Cultures were grown at 378C for 30 min, and then IPTG was added to a final concentration of 0.75 mM for the induction of protein expression (0.75 mM IPTG was chosen on the basis of IBI recommendations and the results of Su et al. [32]). Optical density readings were determined at 600 nm (OD600). Samples were removed from each culture at 0, 30, 60, 90, 120, 150, 180, and 210 min for OD and dot immunoblot analysis. FTNF cultures with and without IPTG induction were monitored for 22 h. Bacterial densities were recorded at 240, 270, 300, 330, 360, 390, 450, 540, and 1320 min. The inducing agent, IPTG (final concentration, 0.75 mM), was added to the FTNF culture that had been grown for 22 h in the absence of IPTG. In a separate study, the growth density curves for FTNF were evaluated following the addition of IPTG to final concentrations of 0.05, 0.10, 0.25, 0.75, 1.5, and 2.0 mM. The FTNF insert was excised from the pFLAG-1 vector that was purified from an uninduced culture. Restriction enzymes, NruI and BglII, were used for the digestion of FTNF and a new pFLAG-1 vector. The insert was ligated into the new pFLAG-1 vector. The transformation of cells and an evaluation of growth curves were performed as described above. Culture supernatants. Following a 60-min exposure of the pFLAG-1 vector cultures and those containing FTNF to 0.75 mM IPTG, the cells were sedimented by centrifugation. The FTNF supernatant was used to resuspend equal densities of unstimulated cells containing only the pFLAG-1 vector. The cell pellets from both cultures were washed three times and resuspended in fresh LB medium with ampicillin. The cell densities were recorded every 60 min during the 240-min incubation at 378C. E. coli (JM101) was grown in the presence of supernatants from cultured cat peritoneal macrophages that (i) had been stimulated with lipopolysaccharide and demonstrated bioactivity and (ii) had been grown in the presence of medium alone and lacked TNF bioactivity (21).

Dot blot analysis. Dot blot analysis was performed on 1 to 5 ml of culture at each time point for both the pFLAG-1 vector and FTNF according to the IBI FLAG instruction manual. Membranes were incubated with 10 mg of anti-FLAG M-1 monoclonal antibody (the IBI antibody directed at the FLAG octapeptide of the fusion protein) per ml. The secondary antibody was biotinylated antimouse immunoglobulin G (1:100,000 in Tris-buffered saline). The blots were then incubated with streptavidin-alkaline phosphatase (1:2,500 in phosphatebuffered saline). Color was developed with nitroblue tetrazolium (Bio-Rad)BCIP (5-bromo-4-chloro-3-indolyl phosphate; Bio-Rad). Rabbit polyclonal antibody raised to the first 12 amino acids in the FTNF sequence (22) was used to identify the TNF protein. The secondary antibody was 1:500 goat anti-rabbit horseradish peroxidase conjugate (Sigma Chemical Co., St. Louis, Mo.), and 10 mg of diaminobenzidine (Sigma) in 20 ml of phosphatebuffered saline and 3 ml of 3% H2O2 were added to develop the color. Evaluation of the fusion protein. Samples containing the bacterial supernatants and cell pellets were collected prior to and at 2 h following IPTG induction of JM101 bacteria containing the FTNF. Purification of the fusion protein was attempted by the method recommended by IBI in the FLAG expression system product manual. This technique failed to release the protein from the cell pellet; therefore, a denaturation-renaturation procedure was employed (16). Sodium dodecyl sulfate–14% polyacrylamide gel electrophoresis (SDS–14% PAGE) was used to evaluate the protein (14). Western blots (immunoblots) were prepared by electrophoretic transfer of proteins to nitrocellulose membranes (33). Screening of TNF activity. A previously described bioassay with mouse WEHI 164 fibrosarcoma cells was used to measure TNF activity (6) in filtered supernatants from E. coli expressing feline, equine, and porcine recombinant FLAGTNF fusion protein. Bioactivity was also tested following the extraction of the feline recombinant protein by the denaturation-renaturation procedure (16). This assay was used to determine TNF activity in feline peritoneal macrophage supernatants (23). Assays were performed on serial dilutions of sample. All samples were assayed in triplicate. Electron microscopy. Cells containing FTNF and cells containing the FLAG

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FIG. 2. Polyacrylamide gel of recombinant feline, porcine, and equine TNF fusion proteins. SDS–14% PAGE demonstrates the presence of an approximately 17-kDa protein following induction of cultures with 0.75 mM IPTG. The first lane contains the Mark 12 (Novex, San Diego, Calif.) molecular weight marker. The second, fourth, and sixth lanes contain the approximately 17-kDa feline TNF fusion protein, the porcine (filled arrowhead) FLAG-TNF, and the 20-kDa equine (open arrowhead) OmpA-FLAG-TNF fusion protein, respectively. Lanes 5 and 7 contain uninduced cells with the genes for porcine TNF and equine TNF, respectively. The fusion protein was confirmed on Western blot with the anti-FLAG M-1 antibody (IBI) (data not shown). vector were collected 2 h after induction with 0.75 mM IPTG. The cells were centrifuged, and the supernatant was discarded. The cells were resuspended in 0.5% glutaraldehyde and embedded in LR White resin (London Resin, London, England) for electron microscopic evaluation.

FIG. 3. Effects of feline TNF expression on bacterial growth. The cell densities (OD600) of E. coli JM101 cells transfected either with the pFLAG-1 vector alone or with the pFLAG-1 vector containing FTNF are plotted over time. Twenty-two hours after induction, the FTNF cultures (postrecovery) were diluted and divided into groups with and without IPTG. The growth of these groups was monitored for an additional 2.5 h. All samples were assayed in triplicate.

CLIN. DIAGN. LAB. IMMUNOL.

FIG. 4. IPTG dose response. The cell densities (OD600) of E. coli (JM101) cells containing FTNF in the pFLAG-1 vector are plotted over time. The cultures were exposed to 0 to 2.0 mM IPTG. All samples were assayed in triplicate.

RESULTS Cultures containing the FTNF gene grew more slowly than those containing only the pFLAG-1 vector (Fig. 1). The induction of FTNF gene expression by the addition of IPTG resulted in a cessation of culture growth within 1 h. During this time, the recombinant FTNF fusion protein was expressed as determined by protein electrophoresis (Fig. 2). Induced cultures of both JM101 (data not shown) and LL308 returned to log-phase growth at approximately 6 h following induction (Fig. 3). Uninduced FTNF cultures reached a maximum density at 3 h. This density decreased until 7 h had passed, at which time normal growth resumed (recovery). The addition of IPTG to these recovered cells did not influence cell density or result in TNF protein expression. The expression of porcine and equine pFLAG-1–TNF genes reduced the growth of JM101 (data not shown) and LL308 E. coli compared with the growth of cultures containing the pFLAG-1 vector alone (Fig. 1B and C), but unlike the results obtained after the expression of feline pFLAG-1–TNF, induction with IPTG did not alter growth curves. Feline, porcine, and equine TNF fusion proteins were confirmed by Western blot assay with anti-FLAG M-1 antibody 2 h following induction with 0.75 mM IPTG. At 22 h of growth, the uninduced and the postrecovery-induced feline cultures failed to express a visible or immunoreactive fusion protein. The filtered supernatants containing the porcine and equine fusion proteins had cytolytic activity in the WEHI assay; however, the feline supernatants lacked bioactivity. IPTG at concentrations of 0.05 to 2.0 mM equally inhibited the growth of E. coli JM101 cells containing FTNF (Fig. 4). Supernatants from IPTG-induced JM101 cells containing the FTNF gene did not inhibit the growth of cells containing the pFLAG-1 vector, suggesting that the toxic factor was not secreted. The washing of JM101 cells induced after 30 min of

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FIG. 5. Effects of washing on toxicity of FTNF. Triplicate samples of JM101 cells containing the empty pFLAG-1 vector or the pFLAG-1 vector with FTNF were grown in the presence of 0.75 mM IPTG for 30 min. The cells were then separated from the supernatant by centrifugation. The cell pellets were resuspended in equal volumes of LB medium containing ampicillin (100 mg/ml) (pFLAG-1 and FTNF) or the supernatant from the FTNF culture (pFLAG-1). Cell densities were measured as OD600 (visible spectrum) every 60 min. Symbols: Ç, FTNF cells washed 30 min after the addition of IPTG; s, pFLAG-1 cells washed 30 min after the addition of IPTG; u, pFLAG-1 cells plus the FTNF supernatant 30 min after the addition of IPTG.

exposure to IPTG did not prevent the inhibition of cell growth in cells containing FTNF. Washing did not alter the growth of JM101 cells containing the pFLAG-1 vector alone (Fig. 5). FTNF was not directly toxic to LL308 E. coli. Fifty microliters of JM101 cells was incubated for 30 min with (i) recombinant human TNF (Genzyme) (20 or 200 U), (ii) 10, 100, or 500 ml of FTNF supernatants containing .2,000 U of TNF activity per ml (from lipopolysaccharide-stimulated, elicited cat macrophages), or (iii) supernatants (10, 100, or 500 ml) from cultured unstimulated feline macrophages. LB medium was added to all samples to achieve a final volume of 1 ml. The cultures were diluted to 1/105, 1/106, and 1/107, and 100 ml from each was plated and incubated overnight at 378C. An evaluation of the plates did not demonstrate a difference in the number of colonies among the treatment groups. The digestion of the FTNF gene from the pFLAG-1 vector in cells with retarded growth (2 h after induction, prior to recovery) and religation of the FTNF gene into a new pFLAG-1 vector resulted in inhibited growth in both the LL308 and JM101 strains of E. coli containing these newly constructed plasmids. The induction of protein expression at 30 or 228C facilitates the expression and secretion of some proteins (27). Varying the induction temperature (37, 30, or 228C) did not eliminate the toxicity of FTNF nor did it result in protein secretion into the supernatant (Fig. 6). The toxicity was associated with the induction of the recombinant protein but was not transferable in the supernatant and was not associated with the addition of naturally occurring FTNF. Prior to induction, the sequence of the spliced genomic FTNF insert was consistent with the TNF coding region of cat genomic DNA. The insert was in the correct reading frame and the flanking region of the vector was intact. The splice sites were confirmed by comparison with the cDNA sequence for FTNF (5). Twenty-four hours following the recovery of cell


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FIG. 6. Polyacrylamide gel of recombinant FTNF induced at 37, 30, and 228C. SDS–14% PAGE demonstrates the presence of a 17-kDa protein (arrow) in the cell pellet, but it is not present in the supernatant of E. coli containing FTNF and induced with 0.75 mM IPTG. The first lane contains the Mark 12 (Novex) molecular weight marker. The third (37p), sixth (30p), and eighth (22p) lanes contain the cell pellets from inductions at 37, 30, and 228C, respectively. Cell supernatants from inductions at 30 and 228C are in the fifth and seventh lanes, respectively. The fourth lane represents a previously confirmed FTNF fusion protein. The TNF fusion proteins were confirmed by Western blotting with the antiFLAG M-1 antibody (IBI) and the rabbit anti-N-terminal FTNF antibody.

growth, the sequences of the insert and the fusion octapeptide were intact. The 59 sense sequencing primer, which originated at the vector tac promoter site, failed to produce the sequence. With an antisense primer originating at the 59 end of the insert, CATSEQ3, the tac promoter region, was sequenced, and an approximately 1,400-base deletion involving a major portion of the promoter site was identified (Fig. 7).

FIG. 7. Deletional mutation of the FTNF–pFLAG-1 vector following recovery of cell growth. The pFLAG-1 vector (lane FLAG), the FTNF–pFLAG-1 vector following the recovery of cell growth (lane REC), and the original FTNF– pFLAG-1 vector (lane FTNF) were digested with BglII (Gibco BRL, Gaithersburg, Md.), a unique restriction enzyme. The digests were subjected to electrophoresis on a 1% agarose gel and stained with ethidium bromide. The first lane contains the 1-kb marker (Gibco BRL). The recovered FTNF–pFLAG-1 vector is approximately 1,400 bases smaller than the vector containing the original FTNF.

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FIG. 8. Electron micrographs of E. coli expressing FTNF or an empty vector. Electron micrographs (magnification, 321,000) demonstrate the presence of inclusion bodies (arrowheads) in the E. coli cells expressing FTNF (A), which are not present in the IPTG-induced E. coli cells containing an empty pFLAG-1 vector (B).

The recombinant FTNF protein was limited to the cell pellet as evidenced by SDS-PAGE and Western blot. A band of approximately 17 kDa was present in the induced cells but not in the supernatant or uninduced cells (Fig. 6). The 17-kDa band was recognized by the anti-FLAG M-1 antibody directed at the fusion protein. An electron microscopic evaluation of E. coli cells expressing recombinant FTNF revealed visible inclusion bodies (Fig. 8). Culture supernatants and extracted (denatured and renatured) recombinant feline protein had no TNF bioactivity. Porcine and equine TNF protein and activity

were present in their respective culture supernatants. Their respective fusion proteins purified according to the FLAG product manual maintained bioactivity. DISCUSSION This study demonstrates that a fusion protein which was toxic to E. coli (JM101 and LL308) was produced by expressing the FTNF gene in the pFLAG-1 vector. The expression of a semilethal gene exerts selection pressure. The result, in this


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TABLE 1. Comparison of amino acids among the known FTNF sequences and with TNF sequences of other species Amino acid in FTNF sequenceb McGraw

Otto

Daniel

Rimstad

Amino acid(s) in TNF sequences other species

R T L A A

R T L T T

W T H T T

W K L A A

W S, R, K L, F, —d T A, T

Amino acida

28 65 75 79 134

Possible effect of mutations (amino acid)c

Unf (R) ND (T) ND (H) Act (A) Act

a

The amino acid number is based on the numbering of the human soluble TNF protein. The McGraw sequence is the original genomic FTNF (17). The Otto sequence is the spliced genomic sequence that produced an inactive protein upon E. coli expression. The Daniel sequence (5) was derived from cDNA, and expression in a baculovirus system produced an inactive protein (1). The Rimstad sequence was a cDNA sequence successfully expressed in E. coli (25). c Unf, protein unfolded; ND, not determined; Act, active protein. d —, not present in bovine sequence. b

study, was the development of a mutant that was unable to express the protein and thereby survived. The mutant identified here contained a deletional mutation of the tac promoter site. While the fusion protein could potentially exert a direct toxic effect on the cells, it is particularly noteworthy that the equine and porcine TNFs in this same system were not toxic (31, 32). The exposure of bacterial cultures to FTNF (from lipopolysaccharide-stimulated, elicited macrophages) did not inhibit their growth. This may have been a result of the inability of the TNF to penetrate the bacterial cell wall or of inadequate inhibitory concentrations of TNF. In vivo, TNF is known to prime macrophages, neutrophils, eosinophils, and natural killer cells to stimulate bacterial killing (7). Direct toxic effects of TNF on parasites and viruses have been recognized, but no direct effect on bacteria has been identified (11). The report of the successful expression of FTNF in E. coli XL-1 blue by Rimstad et al. provides further evidence that the mature TNF protein is not directly toxic (25). These data suggest that the toxic effect of FTNF on E. coli may have resulted from the presence of unique amino acids in this FTNF’s sequence. These amino acids may have led to the formation of insoluble intermediates (18) or altered the tertiary structure, promoting the formation of insoluble aggregates which prevented the release of the protein from the bacterial cell. The threonine found at amino acid 65 in 3 of 4 FTNF sequences (Table 1) (5, 17, 19, 25) is located within the b sheet, which is important in protein folding (12). The lack of activity reported with the expression of the Daniel sequence (1) and the presence of a lysine at this site in the active product of the Rimstad sequence (25) suggest that amino acid 65 might contribute to proper folding and bioactivity. The expression system may be critical to the production of a soluble, active protein. The FLAG system has been successfully employed for the expression of the porcine and equine TNFs (31, 32). The presence of threonine at amino acid site 134 is unlikely to have any effect on activity or folding. Amino acid 134 is represented by threonine in rabbits and in point mutation experiments; conversion of the alanine 134 to threonine had no effect on TNF activity (Table 1). This variation may represent a polymorphism. The mutation of amino acid 28 from a tryptophan to an arginine resulted in the unfolding of the human TNF protein (12). The fusion protein expressed here contained an arginine at amino acid 28. No other known species contains an arginine at this site in the TNF sequence. Two known FTNF cDNA sequences, including one that was successfully expressed as active TNF, coded for a tryptophan at amino acid 28 (Table 1)

(5, 25). The presence of this arginine may represent reverse transcription errors or an instability of the DNA that predisposed it to mutations; however, three different sources of FTNF were sequenced and all contained an arginine at amino acid 28 (17, 19). While only one TNF locus has been identified in the genomes of other species, this has not been confirmed in cats. Multiple loci, variant allelic forms, or the presence of TNF pseudogenes may contribute to the amino acid variations identified. Predicted amino acid differences were also noted between two rabbit TNF cDNAs and the rabbit TNF genomic sequence. These differences were attributed to polymorphisms or errors of transcription (9, 10). Mutations resulting in failure to fold or target the protein appropriately could lead to an accumulation of the fusion protein within the bacteria (29). Protein accumulation interferes with normal cell function and may result in cell death. The overexpression of the protein may result in insoluble aggregates (29). Overexpression of porcine TNF resulted in insoluble but nontoxic aggregates. The induction of expression at 308C slowed protein expression and allowed for an enhanced secretion of the 18-kDa protein (32). In the case of FTNF, unlike TNFs from pigs and horses, the recombinant fusion protein was present in insoluble aggregates within the cell. The recommended extraction procedures (IBI FLAG expression system product manual) failed to release the protein. A denaturation and renaturation procedure (16) was necessary to obtain pure fusion protein. Expression at decreased temperatures did not influence the toxicity of FTNF or allow the secretion of a soluble protein. Several amino acids have been identified that may contribute to the toxic effects of expression of the FTNF fusion protein. Amino acid substitution studies will be necessary to determine the role of each amino acid in toxicity and in the failure to secrete an active protein. Eukaryotic expression of this clone of FTNF is in progress and will help determine if the formation of an insoluble protein is a function of the prokaryotic expression system or of the protein structure. ACKNOWLEDGMENTS We thank Denise Pesti for her technical advice and assistance. This research was supported by a grant from the University of Georgia Veterinary Medical Experiment Station and the Clara B. West Research Foundation. C. M. Otto was an Affiliate Research Fellow of the American Heart Association, Georgia. REFERENCES 1. Beretich, G., A. Carlioz, and W. Tompkins. 1993. Expression of a recombinant feline tumor necrosis factor in a baculoviral system, p. 78. In Research forum. College of Veterinary Medicine, North Carolina State University, Raleigh.

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