J. Biochem. 109, 632-637 (1991)

Expression of cDNA for Batroxobin, a Thrombin-Like Snake Venom

Enzyme

Masahiro Maeda,* Susumu Satoh,* Shingo Suzuki,* Mineo Niwa,*,1 Nobuyuki Itoh,** and

Ikuo Yamashina***

*Product Development Laboratories, Fujisawa Pharmaceutical Co., Ltd., Yodogawa-ku, Osaka, Osaka 532;

**Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto,

Kyoto 606; and ***Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kita-ku, Kyoto, Kyoto 603

Received for publication, November 7, 1990

The cloned cDNA for batroxobin has been expressed in E. coli. Batroxobin could only be

obtained as intracellular aggregates of fusion proteins, fused with a small peptide. To

obtain the mature batroxobin, the recognition sequence for thrombin was inserted between

the peptide and the mature batroxobin. This fusion protein accumulated in an insoluble

form and could easily be purified. After site-specific cleavage of the fusion protein with

thrombin, recombinant batroxobin was isolated by preparative electrophoresis. Batrox

obin with enzymatic activity was obtained by the refolding of recombinant batroxobin.

Batroxobin [EC 3.4.21.29] is a thrombin-like enzyme obtained from Bothrops atrox, moojeni venom. The molecular weight of batroxobin containing six disulfide bonds was shown to be 29,100. In contrast to thrombin, which converts fibrinogen into fibrin by removing fibrinopeptides A and B, batroxobin removes only fibrinopeptide A. The enzyme is currently used clinically for the treatment of thrombotic diseases, since parenteral administration of the enzyme causes the conversion of fibrinogen into a fibrin derivative which is rapidly degraded through a secondary fibrinolytic process and then eliminated via the urine (1, 2).

Batroxobin cDNA and gene have been isolated from a B. atrox, moojeni venom gland cDNA library and the genomic library, respectively. The amino acid sequence deduced from the nucleotide sequence of batroxobin cDNA exhibited significant homology with those of eukaryotic serine proteases, indicating that batroxobin is a member of the serine protease family (3). The exon/intron organization of the batroxobin gene was different from that of the prothrombin gene but very similar to those of the trypsin and kallikrein genes. These results indicate that batroxobin is not a member of the prothrombin family but is one of the trypsin/kallikrein family. Since the snake venom gland is assumed to have originated from the submaxillary gland, it is conceivable that batroxobin belongs to the glandular kallikrein family (4).

Although batroxobin can be purified from venom gland of the snake B. atrox, moojeni, it is difficult to obtain large amounts of batroxobin for studies. We intended to synthesize batroxobin in E. coli by the use of recombinant DNA techniques, which would be suitable for large-scale production. It is known that the recombinant polypeptides pro

duced as cytoplasmic inclusion bodies in E. coli are biologically inactive when the heterogeneous genes are highly expressed, and such inclusion bodies become soluble only after treatments under denaturing conditions (5). Therefore the refolding of the recombinant proteins is necessary to obtain the native protein. However, in cases of proteins containing many disulfide bonds the refolding is often of low efficiency due to inappropriate disulfide bond formation and the final products often show low specific activities.

In this paper, we describe the expression of batroxobin cDNA, the selective cleavage of the fusion protein with thrombin, and successful refolding to obtain biologically active batroxobin.

MATERIALS AND METHODS

Materials-Restriction endonucleases were purchased from Takara Shuzo (Kyoto), Toyobo (Osaka), New England Biolabs (Beverly), and Boehringer Mannheim (Mannheim). T4 DNA ligase and T4 polynucleotide kinase were from Takara Shuzo. DNA polymerase I (Klenow fragment) was from BRL (Gaithersburg). Thrombin was from Mochida (Tokyo).

Strains and Plasmids-E. coli HB101-16 (6) [F-,

recA13, ara-14, proA2, lacY1, galK2, rpsL20 (Smr),

xyl-5, mlt-1, hsdS20 (rs-, mg-), supE44, ă-, TnlO::

htpR16] was used as the host for the expression of genes.

Plasmid pBat-2 (3) (containing the cDNA for batroxobin),

pLS-D1 (7) (containing the synthetic tryptophan promoter, polypeptide LH gene, somatomedin C gene, and fd

phage central terminator), pLS-T2 (7) (containing the

polypeptide LH gene and somatomedin C gene), and

pCLaHtrp3t (8) (containing the polypeptide Cd-LH gene and ƒ¿-hANP gene) were described previously.

Preparation of Synthetic Genes-All oligonucleotides were synthesized by the phosphoamidate method using an automated DNA synthesizer (Applied Biosystems, Model 381A). The synthetic genes for the N-terminus of batroxobin and factor XIII-like polypeptide were constructed by

1 To whom all correspondence and reprint requests should be ad

dressed.

Abbreviations: Bat., batroxobin; SMC, somatomedin C; DTT, di

thiothreitol; PBS, phosphate-buffered saline; BSA, bovine serum

albumin; RIA, radioimmunoassay; HPLC, high-performance liquid

chromatography; ƒ¿-hANP, alpha-human atrial natriuretic poly

peptide.

632 J. Biochem.

Expression of cDNA for Batroxobin 633

the ligation of the uhosnhorvlated oligonucleotides (7. 8).

Construction of Expression Plasmids-The constructions of pBat-SS3 and pBat-SS49 were performed as shown in Fig. 1. The direct expression plasmid, designated as pBat-SS3, was constructed as follows. Plasmid pLS-D1 was digested with BamHI and both cohesive ends were subsequently filled in with DNA polymerase I (Klenow fragment). The linear plasmid was then digested with EcoRI, and the resultant fragment was ligated to the batroxobin gene (RsaI-HpaI fragment from pBat-2) through the synthetic oligonucleotide. The fusion expression plasmid, designated as pBat-SS49, was constructed by ligation of the trp promoter-polypeptide Cd gene (pCLaHtrp3t cleaved with PstI/EcoRI) to the pBat-SS3 cleaved with PstI/Rsal through the synthetic oligonucleotide encoding the factor XIII-like polypeptide.

Two-cistron expression plasmids, designated as pBat-SS5 and pBat-SS8, were constructed as follows. Plasmid pBat-SS5 was constructed by inserting the batroxobin gene (consisting of the synthetic oligonucleotide for the direct expression plasmid and Rsal-HpaI fragment from pBat-2) between trp promoter-SD sequence and somatomedin C

gene of the pLS-D1. Plasmid pBat-SS8 was constructed by ligation of the batroxobin gggggene (PstI-EcoRI fragment from pBat-SS3) downstream to the trp promoter-somatomedin C gene (PstI-EcoRI partially digested fragment from pLS-T2).

All DNA manipulations were carried out essentially as described by Maniatis et al. (9). Constructed plasmids were characterized by restriction endonuclease digestion, and the nucleotide sequence of the synthetic genes was verified by the Maxam-Gilbert method (10).

Expression and Preparation of Recombinant Batroxobin -Bacterial transformants were grown in 400ml of M9

liquid media containing casamino acids (0.5%, w/v) and

ampicillin (25pg/ml) at 30•Ž to an absorbance of 0.6 at 600

nm. 3-Indoleacrylic acid (IAA) was then added to a final

concentration of 10pg/ml to derepress the trp promoter.

Incubation was continued for an additional 2.5h, and the

cells were harvested by centrifugation at 7,000xg for 10

min at 4•Ž. Pellets were suspended in 8ml of phosphate

buffered saline (PBS, pH 8.0) containing 25mM EDTA,

and sonicated for 2 min in an ice bath. After centrifugation

at 18,000xg for 20 min at 4•Ž, the insoluble pellets

(PBS-insoluble fraction) were resuspended in 10ml of 50%

(v/v) glycerol and sonicated, then centrifuged as described

above. The pellets, after being washed with 50% (v/v)

glycerol twice more, were suspended in 4ml of 100mM Tris-HCl buffer (pH8.0) containing 8M urea and 25mM

EDTA and sonicated for 2 min in an ice bath. After

centrifugation at 20,000xg for 20 min at 4•Ž, the super-

natants were collected.

Cleavage of the Fusion Protein with Thrombin-The

solution of partially purified fusion proteins (5ƒÊl) was

treated with thrombin (5 to 8 units) in 40ƒÊl of 10mM

Tris-HC1 buffer (pH 8.0) containing 1M urea, at 37•Ž for 6

h.

Purification and Refolding to Recombinant Batroxobin -Samples digested with thrombin were subjected to

preparative 15% SDS-PAGE (11). Recombinant batrox. obin was separated from the gel by electroelution using Max Yield (Atto, Tokyo) according to the procedure recommended by the manufacturer, and then was precipitated

with four volumes of cold acetone (-20•Ž). After centrifu

gation (-10•Ž, 18,000xg, 20 min), the precipitate was rinsed gently with ice-cold 80% acetone. The precipitate

was dried and then dissolved in 100mM Tris-HCl buffer

(pH8.0) containing 8M urea and 25mM EDTA. This

solution was diluted with a refolding buffer consisting of 50

mM Tris-HC1, 20% glycerol, 0.15 to 1.5M NaCl, 0.1mM

EDTA, and 0.01% BSA (bovine serum albumin). The

solution of recombinant batroxobin was allowed to stand at

room temperature for approximately 20h. The refolded

batroxobin was examined by radioimmunoassy and/or

biological assay (see below).

Radioimmunoassay (RIA) of BatroxobinA 105-fold-

diluted solution (100pl) of anti-batroxobin serum (raised

in rabbit) prepared in the assay buffer (PBS containing

0.5% BSA and 25mM EDTA) and 100pl of 125I-labeled

batroxobin (approximately 105cpm/ml) prepared by the

Iodogen method (12) were added to 500ƒÊl of the renatured

batroxobin solution or to the batroxobin standard solution

in the assay buffer. After incubation at 4•Ž overnight, 100

pl of normal rabbit serum, 100pl of anti-gamma globulin serum (prepared from rabbit) and 900ƒÊ1 of 5% polyethyl

ene glycol 6000 were added. The solution was left standing

at 4•Ž for 2.5h, and then centrifuged at 4•Ž, 3,000xg for

30min. The radioactivity of the pellet was counted with an

auto-gamma-counter (Packard, model 800).

Assay of Enzymatic Activity of Batroxobin on Fibrinogen-

Agarose Plates-Modified fibrinogen-agarose plates (1)

were prepared as follows: 50mg of agarose was dissolved in

5ml of PBS at 90•Ž and the solution was cooled to 45•Ž.

Five milliliters of 0.4% bovine fibrinogen (plasminogen-

free, Miles Scientific, Naperville) in PBS, prewarmed to

45•Ž, was added and the mixture was immediately poured

into a dish and allowed to gel. A 10ƒÊl aliquot of batroxobin

solution was applied to fibrinogen-agarose plates and in

cubated in a humid atmosphere overnight at 37•Ž in order

to cause clot propagation. As batroxobin is capable of

converting fibrinogen to fibrin due to the thrombin-like

activity, the formation of fibrin was observed as distinct

turbidity. The diameter of each clot was measured by

means of a micrometer device and recorded on a semi

logarithmic scale as a function of the batroxobin concentra

tion in the solution.

Other Techniques-In vitro translation was carried out using a Prokaryotic DNA-directed translation kit (Amersham, Tokyo) according to the procedure recommended by the manufacturer. 35S-Labeled proteins were visualized by SDS-PAGE followed by fluorography. HPLC was performed under the following conditions: column, YMC AP-302 (4.6x75mm); detection, absorbance measurement at 210nm; flow rate, 1.0ml/min; elution solvent, 0.1% trifluoroacetic acid with a 10-60% acetonitrile gradient. The amino acid sequence was determined using a gas

phase sequencer (Applied Biosystems, model 470A). The protein concentration was estimated from the absorbance at 280nm assuming that one absorbance unit corresponds to 0.5mg protein/ml.

RESULTS AND DISCUSSION

Construction of the Expression Plasmids and Production of Batroxobin-In order to effect the expression of mature batroxobin, we first constructed the direct expression

Vol. 109, No. 4, 199Vol. 109, No. 4, 1991

634 M. Maeda et al.

plasmid (pBat-SS3, Fig. 1). However, the production of batroxobin was not detectable by SDS-PAGE or radioimmunoassay (data not shown). Further, the translation of batroxobin mRNA was not detected in an in vitro translation system (data not shown).

The construction of the plasmids for the two-cistron

system and fusion protein method is summarized in Fig. 2.

Both pBat-SS5 and pBat-SS8 contain the batroxobin gene and a fused gene made up of somatomedin C gene and a part (corresponding to Cys1-Leu59, designated as polypeptide LH) of the interferon-gamma gene in two-cistron systems downstream from the trp promoter. They are different with respect to the position of the batroxobin gene. Neither pBat-SS5 nor pBat-SS8 produced the mature batroxobin in

Fig. 1. Schematic outline of the construction of the expression plasmids. Batroxobin coding sequences are represented by the open box.

The shaded box represents the synthetic trp promoter region. The double-hatched box represents the factor XIII-like sequence. The hatched

box represents the synthetic fd phage central terminator. Amp' indicates the ƒÀ-lactamase gene. The solid thick line represents the polypeptide

Cd gene, the polypeptide LH, the somatomedin C gene, or the ƒ¿-hANP gene. The thin single line represents the pBR322 sequences. In order

to effect the expression of mature batroxobin, pBat-SS3 was constructed as follows; the 5•L-noncoding region and a part of the coding region for

the signal peptide and zymogen peptide were replaced by the synthetic gene coding for the N-terminal region of muture batroxobin.

Fig. 2. Summary of the structure of constructed plasmids and their capacity for expressing a batroxobin gene. The column headed expression indicates the results from in vitro translation experiments. Translation of protein from each

plasmid is given as detectable (+) or not detectable (-).

J. Biochem.

Expression of DNA for Batroxobin 635

the in vitro translation system (data not shown). However, these plasmids (pBat-SS5 and pBat-SS8) produced fusion proteins, LH-SMC, efficiently with nearly the same yield. The results suggest that the failure of expression of the batroxobin gene is a post-transcriptional event probably due to the formation of secondary structure of the mRNA unsuitable for the translation of batroxobin in E. coli.

Finally we constructed the plasmid which is expected to

encode a fusion protein composed of batroxobin and a part

(Phe23-G1u46, designated as polypeptide Cd) of somatomedin C. This polypeptide has been used effectively as a

partner to form fusion proteins with somatomedin C (7, 13) or ƒ¿-hANP (8). Moreover, to liberate mature batroxobin

from the fusion protein, we introduced an oligonucleotide

corresponding to a peptide with the thrombin recognition

sequence between the Cd and batroxobin genes. Thus the

produced fusion protein possesses the specific thrombincleavage site between the C-terminal of polypeptide Cd and

the N-terminal of the mature batroxobin.

We constructed the expression plasmid for the production of mature batroxobin based on the above considerations (Fig. 1). The peptide contains the cleavage site for thrombin. The synthetic oligonucleotide encoding the tetradecapeptide Met-Asp-Asp-Leu-Pro-Thr-Val-Glu-Leu-Gln-Val-Val-Pro-Arg was inserted between the polypeptide Cd gene and the mature batroxobin gene. The resulting expres-

Fig. 3. SDS-PAGE analysis of E. coli lysates. E. coli HB101.16 containing pBat-SS49 (lanes 1 and 2) was grown under inducing conditions, and then was lysed by sonication. Lane 1, cellular soluble proteins; lane M, molecular weight markers (top to bottom, 92,500, 66,200, 45,000, 31,000, 21,500, 14,400); lane 2, cellular insoluble proteins. The arrow indicates the fused batroxobin containing factor XIII-like sequence

present in the PBS-insoluble fraction. For SDS-PAGE, 15% gel was used.

Fig. 4. Cleavage of the fusion

protein produced from E. coli

harboring pBat-SS49 with

thrombin. The solubilized fusion

protein (P-SS49) was cleaved

with different concentrations of

thrombin in various concentra

tions of urea. Cellular insoluble

proteins were washed three times

with 50% (v/v) glycerol, dissolved

in 8 M urea, 100mM Tris-HCl,

pH 8.0, 25mM EDTA, and then

centrifuged at 20,000xg for 20

min. The supernatants (5ƒÊ1) were

treated with thrombin in 40ƒÊ1 of

10mM Tris-HCl buffer (pH 8.0),

at 37•Ž for 6h. Lane M shows

molecular weight markers (top to

bottom, 92,500, 66,200, 45,000,

31,000, 21,500, 14,400).

sion plasmid, designated as pBat-SS49, was used to transform E. coli HB101-16. The bacterial transformants were cultivated in M9 liquid media containing casamino acids to produce the fused batroxobin (Fig. 3, approximately 40mg of the fused batroxobin was produced in 1 liter of culture broth). The produced fusion polypeptide was aggregated to form inclusion bodies (5) which were clearly seen by the use of a phase-contrast microscope (data not shown).

We constructed other expression plasmids to produce

fusion proteins using the synthetic genes encoding poly

peptide LH or polypeptide Cd-LH (described previously;

7, 8). When fused with these small peptides batroxobin was

produced in sufficient amounts. The reason why the batroxobin could only be expressed as a fusion protein, is unclear.

It is suggested that the fused polypeptide genes (poly

peptides LH, Cd, and Cd-LH) change the environment of

the 5•L end of the mRNA and this causes disruption of the

stable secondary structure. Further the fused polypeptides

may be able to stabilize batroxobin in E. coli.

Isolation and Purification of Batroxobin Generated from

Fig. 5. SDS-PAGE analysis (A) and HPLC profile (B) of the recombinant mature batroxobin purified by preparative elec-trophoresis. For SDS-PAGE, 15% gel was used. HPLC conditions are described in "MATERIALS AND METHODS."

Vol. 109, No. 4, 1991

636 M . Maeda et al.

Fig. 6. Refolding of the recombinant batroxobin. (A) A solution of the purified mature batroxobin (500kg/ml) was diluted to several concentrations (2.5-25pg/ml) with dilution buffer consisting of 50mM Tris-HCl, pH 8.0, 150mM NaCl, 20% glycerol, 0.1mM EDTA, and 0.01% BSA, and then the solution was left standing at room temperature for about 20 h. Refolding was followed by assaying batroxobin with RIA. (B) The solution containing 2.5kg of batroxobin in 1 ml was adjusted to pH 6-10.5 with 1M HCl or 1M NaOH and refolded at room temperature for about 20h. (C) The same concentration of batroxobin was adjusted to various NaCl concentrations in the range of 0-1.5M.

the Fusion Protein-The fused batroxobin was refolded in

the redox buffer which contained reduced glutathione and

oxidized glutathione (14). However, the refolded protein

exhibited no reactivity towards the specific antibody (data

not shown). Therefore, the refolding was applied to the

mature batroxobin obtained from the fused polypeptide.

Thus, the fusion protein synthesized from pBat-SS49,

designated as P-SS49, was treated with thrombin in 10 mM

Tris-HC1 buffer (pH 8.0) containing 8, 4, 2, and 1 M urea,

respectively, at 37•Ž for 6h (Fig. 4). P-SS49 could be

completely digested with 10 units of thrombin per 40ƒÊl of

the reaction mixture containing 1M urea. On purification of

mature batroxobin by preparative electrophoresis (the

recovery of mature batroxobin from the polyacrylamide gel

was about 70%), the cleavage product was obtained as a

homogeneous preparation, as shown by SDS-PAGE analy

sis (Fig. 5). The amino acid sequence from the N-terminal

of the purified protein was determined. The sequence,

Val-Ile-Gly-Gly-Asp, corresponded to that of the native

Fig. 7. Detection of the biological activity of refolded recom-

binant mature batroxobin on a fibrinogen-agarose plate. A 10ƒÊ1

sample of the refolded batroxobin in the modified dilution buffer was

applied to a fibrinogen-agarose plate and incubated overnight at 37•Ž.

mature batroxobin (3).These results show that the cleavage with thrombin

occurred at the expected specific site. All the recombinant

proteins containing the factor XIII-like sequence were correctly cleaved with thrombin irrespective of the type of the polypeptides fused (polypeptides LH, Cd, or Cd-LH)

(data not shown). It seems that thrombin recognized and cleaved the factor XIII-like sequence of the fusion protein faster than it might cleave the putative recognition sequences for thrombin located in the batroxobin sequence. Most likely, the insertion of 14 amino acid residues containing the factor XIII-like sequence may have contributed to our success. This approach may be applicable to other large-molecular recombinant fusion proteins containing many lysine and arginine residues.

Refolding of the Recombinant Batroxobin Produced in E. coli-We tried to refold the recombinant protein so as to obtain bioactive batroxobin. Although there are no established refolding methods for recombinant proteins produced in E. coli up to the present, several methods have been used to refold many recombinant proteins. Solubilization of recombinant proteins in buffer free of denaturing agents was achieved by dialysis or by dilution. The efficiency of this renaturation process may depend on such factors as the concentration and purity of the recombinant polypeptide, pH, ionic strength of the medium and presence or absence of thiol reagents (Refs. 15 and 16 for reviews). We examined several methods (data not shown). We selected Hagers' method (17) for the refolding of the recombinant batroxobin, as the refolding efficiency was comparatively high.

Refolding of the recombinant batroxobin in the dilution buffer without a thiol reagent such as dithiothreitol (DTT) was firstly tried. The refolding efficiency was increased about 20-fold compared to that in the presence of DTT. Since the recombinant mature batroxobin had been purified by preparative SDS-PAGE under reducing conditions, the

J. Biochem.

Expression of cDNA for Batroxobin 637

thiol reagent might not be necessary. In the following

experiment, this dilution buffer without DTT was used. The

refolding efficiency of batroxobin was highly dependent on

the concentration of the recombinant batroxobin, pH and

ionic strength of the medium: the optimum conditions for

the refolding of recombinant batroxobin were a concentra

tion of 1-5ƒÊg/ml, pH 6.5-8.5, and 0.5-1.3M NaCl, pH

7.5, as shown in Fig. 6.

The biological activity of the recombinant batroxobin was assayed by measuring its ability to convert fibrinogen to fibrin. It showed thrombin-like activity on a fibrinogenagarose plate (Fig. 7). The specific activity (biological activity/immunoreactivity assayed by RIA) of the batroxobin derived from the fusion proteins was 60-84% of that of mature batroxobin. The lower specific activity may be because partially refolded batroxobin with no or little biological activity is recognized by polyclonal antibodies. The low recovery (about 3%) of batroxobin activity as a result of the low efficiency of the refolding is a problem which remains to be solved.

These findings provide a basis for producing a large

amount of the recombinant protein. The availability of

large quantities of bioactive batroxobin produced in E. coli

will make possible a number of interesting biochemical

experiments.

We thank Drs. Y. Saito and M. Kobayashi of our group for helpful

discussions and T. Tamura in our laboratories for amino acid sequence analysis.

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