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ISSN 0582-9879                                          ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(1): 6-10                                       CN 31-1300/Q

　

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-Dong１, 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 Ser206 [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 Cys31-165,  Cys52-68,  Cys100-258,  Cys144-212,  Cys176-191,  and Cys202-237.  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%.

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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, yulun@mail.dlptt.ln.cn