Cloning,
Expression and Purification of Gussurobin,  A Thrombin-like Enzyme from the Snake Venom of Gloydius
ussuriensis

YANG
Qing, HU Xue-Jun, XU Xiao-Ming, AN Li-Jia^*, YUAN Xiao-Dong^１, SU Zhi-Guo²

( Bioengineering
Institute, Dalian University of Technology, Dalian, China；

¹ Takara Biotech
Dalian Co.  Ltd.,  Dalian 116600, China;

² The State Key
Laboratory of Biochemical Engineering, 
Institute of

Chemical Metallurgy, the
Chinese Academy of Sciences, Beijing 100080, China )

JANSON Jan-Christer

( Biosurface Science
Center, Biomedical Center 577, 75 123 Uppsala, Sweden )

Abstract    Total RNAs were extracted from the venom gland
of the snake Gloydius ussuriensis. 
The cDNA of gussurobin,  a
thrombin-like enzyme from Gloydius ussuriensis,  was cloned and amplified by RT-PCR.  Assay of the nucleotide sequence of the
cDNA allowed postulation of the complete amino acid sequence for Gloydius
ussuriensis,  Chinese Viperdae.  Its amino acid sequence exhibits
significant homology with that of other snake thrombin-like enzymes.  The cDNA of gussurobin was inserted
into the vector pPIC 9K and expressed successfully in Pichia pastoris,  strain GS115. The recombinant
gussurobin was separated and purified from 500 ml culture and showed one band
on SDS-PAGE.

Key words      thrombin-like enzyme; 
gussurobin;  cDNA
sequence;  expression;  purification

More than 200 species of
venomous snakes are now known on the earth.  They are classified into four major families:  (1) Hydrophiae,  (2) Elapidae,  (3) Viperdae and (4) Crotalidae^[1].  Thrombin-like enzymes,  as a family of serine-protease,  are widely distributed among the venom
of Viperidae and Crotalidae families^[1].  In contrast with thrombin,  which converts fibrinogen into fibrin
by splitting off fibrinopeptides A and B, 
the thrombin-like enzyme only splits off fibrinopeptide A.  They have been extensively studied both
by basic researchers and clinicians because of potential therapeutic use in
myocardial infarction and thromobotic diseases since 1970’s^[2―4].
 

So far five thrombin-like enzyme preparations have
been commercially available,  they
are ancrod [US patent 4, 585, 653], 
batroxobin,  reptilase,  crotalase,  and thrombin-like enzyme from Agkistrodon contortrix^[5].  However,  the popularized clinical use of thrombin-like enzymes has
been limited by:  (i) immunologic
reactions in patients,  most
probably due to trace contaminants in the commercial preparations,  and (ii) limited availability of the
snake venom,  and thus (iii) high
price to cause too much economical burden to patients^[6].  Production of thrombin-like enzyme or a
derivative by recombinant technology is a way to cope with the above
problems.  Recently,  the methylotrophic yeast Pichia
pastoris has been developed into a powerful expression system for a number
of foreign genes^[7―9]. 
This expression system has several important characteristics.  It can grow conveniently to high cell
density in an ordinary medium;  it
can secrete proteins under the control of the efficient and tightly regulated
promoter of the alcohol oxidase gene (AOX1);  and most important, 
it can produce proteins with correct folding and post-translational
modification^{[10, 11]}.  

Gussurobin [EC 3.4.21.7] is a thrombin-like enzyme
from the venom of Gloydius ussuriensis (Chinese Viperdae).  So far neither its amino acid sequence
nor cDNA sequence has been reported yet. 
This paper reports the cloning of cDNA of  gussurobin and its successful expression in Pichia pastoris,  strain GS115.

 

1  Materials
and Methods

1.1  Isolation of total RNA from snake venom

The snake Gloydius ussuriensis was
sacrificed.  Its poison gland was
removed and immediately homogenized by adding Trizol Regent (Gibco BRL,  USA).  4 μg of total RNA was extracted according to
the method described by the manufacturer.

1.2  Synthesis and amplification cDNA by
RT-PCR

1 μg of total RNA ( containing 4―5 ng of mRNA) was incubated at 65 ℃ for 10 min and then transferred on ice
immediately.  Synthesis of cDNA was
conducted  according to
manufacturer’s protocol.  RT-PCR
kit (5′ -Full RACE Core Set) developed by Takara
company was used for RT-PCR to obtain the full length nucleotide sequence.  Two primers,  TLE-F1 and TLE-M13M4 (provided by the Takara company),  were used (see Fig.1).

TLE-F15′-ATGGTGCTGATCAGMGTG-3′(M=A+T)

TLE-M13 M45′-CGCCAGGGTTTTCCCAGTCACGAC-3′

Fig.1  Sequence of primers for RT-PCR

1.3  Sequencing of the newly synthesizd cDNA
of gussurobin

The nucleotide sequences were assayed by the dideoxy
chain-termination method using the “walking
primer” strategy (see Fig.2).  M13 universal and synthetic oligonucleotide
(Takara Biotech,  Japan) were
applied as 3′ and 5′ terminal
primers,  respectively.

Fig.2  Sequence of primers and “walking strategy” for sequencing

1.4  Construction and screening for multiple
inserts

The mature gene of gussurobin was amplified by PCR to
place EcoRI site at its 3′ terminal
and place a-factor signal sequence ending at cleavage
site of

Ste13 at its 5′ terminal
by using primers 5′-GAAAA-GAGAGGCTGAAGCTATCATTGGAGGTGATGA-ATG-3′ and 5′-CCGAATTCTTATCATGGGGGG-CAGGTTGCA-3′.  This
fragment of gussurobin  gene was
inserted into expression vector pPIC 9K (Invitrogen,  USA).  Purified
construct was linearized by restriction enzyme SalI (Takara Biotech,  Japan) and integrated into yeast
genome,  strain GS115,  by electroporation (Bio-Rad Gene
Pulser,  USA).Transfermants were
firstly selected by their ability to 
grow on minimal media MM / MD and then selected by their resistance to
G418. Single colony with phenotype of His⁺Mut⁺ and higher
resistance to G418 was finally obtained.

1.5  Expression and purification of
recombinant gussurobin

Selected transfermants were pregrown at 30 ℃ in 0.5 L baffled shake flasks containing 0.15 L rich
medium (20 g/L tryptone,  13.4 g/L
yeast nitrogen base,  4 × 10^-4 g/L biotin,  1 % glycerol, 
0.1 mol/L potassium phosphate buffer,  pH 6.0).  The
cells were grown for 24 h with an additional 1 % glycerol after 12 h.  The culture was centrifuged and the
pellet containing the cells was resuspended in rich medium with methanol (20
g/L tryptone,  13.4 g/L yeast
nitrogen base,  4 × 10^-4 g/L biotin,  1 % methanol, 
0.1 mol/L potassium phosphate buffer,  pH 6.0).  After
induction for 72 h,  the culture
was collected and adjusted pH to 5.0 and then centrifuged to remove cells and
precipitates.  Exchanging buffer to
20 mmol/L methylpiperazin-HCl,  pH
5.0,  by using Sephedex G 25,  the supernatant was applied to
Q-Sepharose Fast Flow (Pharmacia Biotech AB,  Sweden) and the target protein was eluted at the gradient of
0―0.5 mol/L NaCl in 20 mmol/L
methylpiperazin-HCl,  pH 5.0 in 20
column volume.  The activity peak
was pooled and then applied to Phenyl HP (Amersham Pharmacia Biotech AB,  Sweden) in a start buffer of 1 mol/L
ammonium sulfate in 20 mmol/L Tris-HCl, 
pH 7.2. The active fractions collected were concentrated by
ultralfiltration (Amicon,  USA) up
to 5 times and 0.5 ml of them was applied onto Superdex 200 column.  The active material obtained was loaded
to ConA Hitrap (Pharmacia Biotech AB, 
Sweden),  eluted by 0.5
mol/L NaCl and 0.5 mol/L methyl- a
-D-glycopyanoside in 20 mmol/L Tris-HCl, 
pH 7.4.

1.6  Amidolytic activity assay

Amidolytic activity was assayed using chromogenic
substrate (N-a-p-tosyl-Gly-Pro-Arg-p-nitroanilide,  Sigma company)^[12].  One unit of amidolytic activity was
defined as the amount of enzyme necessary to hydrolyze 1.0 mmol of substrate per min.  Ancrod,  commercially available from Sigma,  was used as standard enzyme.  

1.7  Arginine esterase assay

Arginine esterase activity was measured as described
by Yabuki et al^[13], 
using N-p-tosyl-L-arginine methyl esterase (TAME,  Sigma , USA)as substrate.

1.8  Protein concentration

Protein concentration was determined by the method of
bicinchoninic acid^[14].

1.9  SDS-PAGE electrophoresis

All manipulations of
electrophoresis were performed according to the manual using PhastGel System
(Pharmacia Biotech AB,  Sweden).

2  Results and
Discussion

2.1  Nucleotide sequence analysis of
gussurobin

The RT-PCR product,  confirmed by agarose gel electrophoresis analysis to carry
the original genetic information, 
was subjected directly to cDNA sequence analysis.  Besides the 5′ and 3′ terminal primers,  several intermediate primers were
synthesized to release complete sequence of cDNA.  Thus a 783 bp nucleotide was obtained (Fig.3).  The translation-initiation site was
assigned to the first methionine codon, 
ATG (nucleotides 1―3), 
and termination codon TGA was found at nucleotides 781―783. According to Nielsen et al^[15],  the putative prepeptide and propeptide
comprised amino acid residues 1 through 18,  and 19 through 24. The restriction enzyme site was located
at residue 18. Based on homology^[16―20],  we could deduce the
catalytic amino acid residues to be His⁶⁷,  Asp¹¹², 
Asp²⁰⁰ and Ser^{206 [15]}.  Moreover, 
gussurobin contained 12 cysteine residues and we could predict that each
cysteine residue contributed to form disulfide bond.  The six disulfide bonds of gussurobin should appear to be
Cys^31-165,  Cys^52-68,  Cys^100-258,  Cys^144-212,  Cys^176-191,  and Cys^202-237.  Although most of snake thrombin-like
enzymes are known to have two sites for glycosylation (Asn-X-Thr),  gussurobin has only one possible
glycosylation site,  Asn¹²⁴-Ser¹²⁵-Thr¹²⁶
(Fig.3).

Fig.3  The nucleotide sequence of gussurobin
cDNA and the deduced amino acid sequence of the gussurobin

2.2  Expression and purification of
gussurobingussurobin

According to several reports of successful expression
in E. coli^[16―19], 
we have also tried to express gussurobin in E. coli under T7
promoter with a 6-His tag. 
However,  by this way,  there was no target protein to be
retained by the affinity column which was expected to entrap histidines.  Hahn et al^[20]
reported the same result when they tried to express batroxobin,  a thrombin-like enzyme from Bothrops
atrox,  Moojeni venom.  The reason why some kinds of
thrombin-like enzymes can be expressed in E.coli,  while others can not,  is not clear yet,  though these enzymes exhibit extremely
high homology in cDNA sequence.  

Finally,  we chose to use yeast expression
system.  The transfermants with
highest resistance to G418 (4 g/L in YPD) was applied to study expression of
gussurobin in 500 ml shaking flask. 
Having been inducted with methanol for 36 hours,  the target enzyme could be examined in
the supernatant by measuring amidolytic activity.   Compared with minimal medium,  rich medium led to higher expression
level of target protein.  The
higher yield was probably due to two reasons.  One reason is that cells can grow better in more nutritious
medium,  and the other reason,  which maybe most important to
gussurobin,  is that peptides in
the medium tend to inhibit protelytic activity and thus could protect
targetprotein.

The recombinant enzyme in the supernatant was  separated and purified by the following
4 steps sequentially involving: 
Q-Sepharose FF,  Phenyl
HP,  Superdex 200 and ConA
(Fig.4).  Table 1 showed the
recovery and efficiency of each purification step.  The purified recombinant gussurobin showed one band on
SDS-PAGE (Fig.5).

Fig.4 
Separation and purification of recombinant gussurobin by column
chromatography

(A) 
Q-Sepharose FF;  (B)  Phenyl HP;  (C)  Superdex
200;  (D)  Affinity ConA. 
Experimental conditions in detail, 
see section  “Materials and Method”.

Fig.5  SDS-PAGE of recombinant gussurobin

1,  1 g/L of purified recombinant
enzyme;  2,  marker.

The recombinant enzyme,  specific binding to the affinity
adsorbent――ConA, 
indicates it is a glycoprotein ［Fig.4(D)］.  The
molecular mass of this enzyme is about 28 kD according to SDS-PAGE
(Fig.5).  Compared with the
theoretic molecular mass calculated by adding all amino acids,  26 000,  the difference is probably contributed by the sugar
chain.  In this case,  the percentage of the sugar moiety is
about 7%.

References

1 
Kim YS,  Hahn BS,  Kim WS,  Chang IM. 
Biochemical and pharmacological properties of thrombin-like protein from
Agkistrodon calliginosus. 
J  Toxicol Sci,  1998,  23 (Supplement II):  218―223

2 
Herzig RH,  Ratnoff OD,  Shainoff JR.  Studies on a procoagulant fraction of Southern copperhead
snake venom:  the preferential
release of fibrinopeptide B.  J
Lab Clin  Med,  1970,  76(3): 
451―465

3 
Itoh N,  Tanaka N,  Funakoshi I,  Kawasaki T, 
Mihashi S,  Yamashina
I.  Organization of the gene for
batroxobin,  a thrombin-like snake
venom enzyme.  Homology with the
trypsin / kallikrien gene family.  J
Biol Chem,  1988,  263(16):  7628―7631

4 
Markwardt F ed.  Handbook of
Experimental Pharmacology, 
Vol.46,  Berlin:  Springer-Veröag,  1978,  451―481

5 
Latallo ZS.  Retrospective
study on complications and adverse effects of treatment with thrombin-like
enzymes――a
multicentre trial.  Thromb
Haemost,  1983,  50:  604―609

6 
Burkhart W,  Smith GF,  Su JL,  Parikh I, 
LeVine H 3rd.  Amino acid
sequence determination of ancrod, 
the thrombin-like α– fibrinogenase from the venom of Akistrodon rhodostoma.  FEBS Lett,  1992,  297(3): 
297―301

7 
Yang JY,  Hui JY,  Li GD,  Wang Y, Yuan HY, Li YY.  Expression of the recombinant hepatitis B virus surface
antigen carrying PreS epitopes in Pichia pastoris.  Acta Biochim Biophys Sin,  2000,  32(2)
:  139―144

(引自：  生物化学与生物物理学报)

8 
Xin L,  Zhang L,  Xu R,  Zhang Q,  Ye
Q,  Li ZP,  Gan RB.  Expression of human angiostatin in Pichia pastoris and
the detection of its anti-angiogenic activity.  Acta Biochim Biophys Sin,  2001,  33(3)
:  291―295

9  Ma
L,  Wang XN,  Zhang ZQ,  Zhou XM,  Zeng
GF,  Chen AJ.  Expression,  purification and biological activity analysis of human vascular
endothelial growth factor (VEGF₁₆₅) in Pichia pastoris. 
Acta Biochim Biophys Sin,  2001,  33(3) :  325―330

10  Cregg
JM,  Vedvick TS,  Raschke WC.  Recent advances in the expression of foreign genes in Pichia
pastoris.  Biotechnology,  1993,  11:  905―910

11 
Borgheresi RA,  Palma
MS,  Ducancel F,  Camargo AC,  Carmona E. 
Expression and processing of recombinant sarafotoxins precursor in Pichia
pastoris.  Toxicon,  2001,  39:  1211―1218

12 
Lottenberg R,  Christensen
U,  Hackson CM,  Coleman PL.  Assay of coagulation protease using peptide chromogenic and
fluorogenic substrates.  Method
Enzymol,  1981,  80:  341―361

13 
Yabuki Y,  Oguchi Y,  Takahashi H.  Purification of kininogenase from the venom of Agkistrodon
caliginosus (Kankoku-mamushi). 
Toxicon,  1991,  29:  73―84

14 
Smith PK,  Krohn RI,  Hermanson GT,  Mallia AK, 
Gartner FH,  Provenzano
MD,  Fujimoto EK et al.  Measurement of protein using
bicinchoninic acid.  Anal
Biochem,  1985,  150:  76―85

15 
Nielsen H,  Engelbrecht
J,  Brunak S,  von Heijne G.  Identification of prokaryotic and eukaryotic signal peptides
and prediction of their cleavage sites. 
Protein Eng, 
1997,  10:  1―6

16 
Pan H,  Zhou YC,  Yang GZ,  Wu XF.  Clonging
and expression of cDNA for thrombin-like enzyme from Agkistrodon halys
Pallas snake venom.  Acta
Biochim Biophys Sin, 
1999,  31(1) :  79―82

17 
Au LC,  Lin SB,  Chou JS,  Teh GW,  Chang
KJ,  Shin CM.  Molecular cloning and sequence analysis
of the cDNA for ancrod,  a thrombin-like
enzyme from the venom of Calloselasma rhodostoma.  Biochem J,  1993,  294(Pt2):  387―390

18 
Itoh N,  Tanaka N,  Mihashi S,  Yamashina I.  Molecular
cloning and sequence analysis of cDNA for batroxobin,  a thrombin-like snake venom enzyme.   J Biol Chem,  1987,  262(7): 
3132―3153

19 
Deshimaru M,  Ogawa T,  Nakashima KI,  Nobuhisa I.  
Accelerated evolution of crotalinae snake venom gland serine
proteases.  FEBS Lett,  1996,  397:  83―88

20      
Hahn BS,  Yang KY,  Park EM,  Chang IM,  Kim
YS.  Purification and molecular
cloning of calobin,  a thrombin-like
enzyme from Agkistrodon caliginosus (Korean viper).  J Biochem,  1996,  119:  835―843

Received:  June 26, 2001Accepted:  August 28, 2001

This
work was supported by the grant No. 99305001 from Liao-ning Province Scientific
Techonogy Fund Program and the Foundation of NUTEK by Swedish Government

^* Corresponding author: 
Tel, 86-411-3682476;  Fax,
86-411-3685241;  e-mail,
[email protected]