BIOTECHNOLOGY TECHNIQUES Volume 8 No.3 (March 1994) pp.1 79-182 Received as revised 3rd February

PURIFICATION AND CHARACTERIZATION OF RECOMBINANT

HUMAN PRO-UROKINASE PRODUCED IN SILKWORM

USING A BACULOVIRUS VECTOR

Yin Gao Meihao Hu’ Department of Biology, Peking University, Beijing 100871 , China

SUMMARY

A recombinant baculovirus (BmNPV-pk2) was constructed by inserting the human pro-urokinase(proUK) cDNA into the genome of baculovirus Bombyx mori

nuclear polyhedrosis virus (BmNPV > adjacent to the strong polyhedrin promoter. The recombinant virus replicated in silkworm larvae, which synthesized 3Opg pro- UK/ml in the haemolymph within 4 days post-infection. Purification to near homog- eneity was accomplished by fractional precipitation with ammonium sulphate and im- munoaffinity chromatography with an overall yield of 23% and a specific activity of 10O,OOOIU/mg in fibrin plate assay. This purified product was comprised of a single chain protein with approximately Mr. 50kDa as determined by SDS-PAGE gel. The N-Terminal amino acids sequence revealed that the secretion signal of pro-UK was correctly processed.

INTRODUCTION

Pro-urokinase or pro-UK, the single chain urokinase-type plasminogen activa- tor , is a glycoprotein of 4 11 amino acids with a Mr. of approximately 54kDa(Homes et al. , 1985). Cleavage of the I,ys158-Be159 peptide bond results in conversion into

a two-chain derivative (urokinase or UK). It has been reported that the single-chain form had a higher specific thromobolytic activity and better selectivity for fibrin than the two chain form when tested in vitro(Sumi et al. , 1983). The single-chain form was purified from urine(Husain et al. , 19831, plasma (Wun et al. , 1982), kidney

cells(Sumi et al. , 1982) and certain tumor cells (Wun et al. ,1982). The recombinant pro-UK was produced in E. coli (Homes et al. , 1985)) Chinese hamster ovary cells (Nells et al. , 1987) , COS- 1 cells (Chcng et al. , 1988) and in insect cells (K ing et al. ,199l). We have expressed human pro-UK at high level using BmNPV Silkworm expression system (Maeda et al. ,1985) and purified it by immunoaffinity chromatog- raphy. The silkworm is easily mass-cultured and an abundant supply of insect’ hosts can be provided at low cost.

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MATERIAL AND METHODS

Construction of trassf er vector p BF d-PTO-u # :

Polyhedrin-fusion vector pBF4 ,obtained from Susumu Maeda(University of Cal- ifornia) ,was used as a transfer vector. Human pro-UK cDNA was cloned by our lab. (Hu, 1993). Transfer vector pBF4-pro-UK was constructed by insertion of pro-UK cDNA cut off from pBluescript-pro-UK by Xba I into the multiple cloning site of pBF4. The correct insertion and orientation with respect to polyhedrin gene was confirmed by restriction map and DNA sequencing.

1&aG~n and purification of recombinasl m+us:

Bm-N cells and wild-type virus BmNPV-C6 were provided by Ruiyin Chu (Peking University >. Plasmid pBF4-pro-UK DNA, together with wild-type BmNPV DNA, was introduced into cultured Bm-N cells by co-transfection using the calcium phosphate co-precipitation method (Summer and Smith, 1987 >. Re- combinant virus BmNPV-pk2 was identified and purified from a viral stock from the transfection by four rounds of plaque purification.

Purifzkalk?n of p~o-fjI< from silkworm haemolymph:

Haemolymph of silkworm larvae was collected four days post-infection by piercing with a pin and squeezing slightly near the larvae body abdominal appendage. The protein product form haemolymph was purified by fractional precipitation with 0. 2-O. 4s ammonium sulphate and by immunoaffiniry chromatography(Nells et al. , 1987). The immunoaffinity chromatography column was made by coupled mono- clonal antibody to activated sepharose 4B. Monoclonal antibody was generously pro- vided by Roger H. Lijnen and Desire Collen (University of Leuven , Belgium).

N-Termtital amko acids sequence awalysis: N-Terminal amino acids sequence analysis was performed using automated Ed-

man degradation (Edman and Begg , 1962) on an Applied Biosystems 477A protein/ peptide Sequencer and 120A amino-acid Analyzer.

Specific fibrinolytic activity of pro-UK was measured on fibrin plates by compar- ison with the International Reference Preparation for Urokinase ( Astrup and Mullertz, 1952). The content of pro-UK was determined by ELISA.

RESULTS AND DISCUSSION

IsoLnLioir of recom.btiaarct I?~TUS RmNPV-pk2:

pBF4-pro-UK plasmid DNA and the BmNPV genomic DNA were cotransfccted

into Bm-N cells and a stable recombinant virus BmNPV-pk:! was isolated by plaque

assay on Bm-N cells. The recombinant virus was confirmed by Southern blot of the

recombinant virus DNA with pro-UK cDNA as a probe.

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When cultured Bm-N cells and silkworm larvae were infected with the re-

combinant virus BmNPV-pk2, the Bm-N cells showed typical cytopathic effects 2

days after infection, the expression levels of pro-UK 4 days post infection were

3. lug/ml(106 infected cells) in the cells fluid and 3Oug/ml in the infected silkworm

larvae haemolymph determined by ELISA.

Fig. 1 SDS-PAGE of pro-urokinase expressed in silkworm larvae on 12% gel lane1 :protein markers. lane2 rurokinasc purified from human urine, non-reduced. lanes: pro-urokinaqe puriFied from haemolymph of silkworm larvae infected with recombinant

virus BmNPV-pk2, reduced with dithiothreitol.

About 25ml haemolymph from 80 silkworm larvae infected with BmNPV-pk2

was collected. After purification according to Methods, 0. 17Omg pro-UK was

collected, the yield was 23%. The purified product had a specific activity of

lOO,OOOIU/mg on fibrin plate and appeared in a single band with a Mr. of approx-

imately 50 kDa as determined by SDS-PAGE gel(Fig. 1). Western blotting indicated

that labelled antibody to pro-UK reacted exclusively with the major band. N-Ter-

minal amino acids sequence analysis of first five amino acids (Scr-Asn-Glu-Leu-His)

were identical to those of human pro-UK.

We have shown that pro-UK has been highly expressed in silkworm using a bac-

ulovirus vector. The result of N-Terminal amino acids sequence analysis showed that

the signal sequence for the secretion of pro-urokinase was recognized and cleaved at

the correct site in the silkworm, and indicated that the fusion peptide didn’t affect

signal sequence cleavage. The purification of pro-UK from silkworm larvae

hacmolymph is simple and effective. The silkworm may be useful for mass-produc-

tion of foreign gene product.

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REFERENCES

Astrup,T. and Mullertz,S. (1952) Arch. B&hem. Biophys. 40, 346-351. Cheng ,S. M. , Lee ,S. G. , and Kalyan,N. K. (1988) Gene 69, 357-363. Edman ,P. and Begg ,G. (1962) Eur. J. Biochem. 1, 80-91. Homes,W. E. , Pennica,D. , Blaber ,M. , Rey,M. W. , Guenzler , W. M. ,

Steffen , G. J. , and Heyneker , H. L. (1985) Bio/Technology 3, 923-929. HU PM. (1993) Acta Scientiarum Naturalium Universitatis Pekinensis 29, 209-

214. Husain, S. S. , Gurewich,V. , and Lipinsik , B. (1983) Arch. Biochem.

Biophys. 220, 31-38. King,L. A. , Kaurk,K. , Mann ,S. G. , Lawire ,A. M. , Steven, J. , and

Ogden,J. E. (1991) Gene 106, 151-157. Maeda ,S. , Kawai ,T. , Obinata,M. , Fujiwara ,H. , Horiuchi ,T. , Saeki ,Y. ,

Sato,Y. , and Furasawa,M. (1985) Nature 315, 592-594. Nells,L., Lijnen, H. R. , CoLlen, D. , and Holmes, W. E. , (1987) J. Biol.

Chem. 262, 5682-5689. Sumi, H. , Kosugi,T. , Matsuo,O. , Mihara,H. , and Toki, N. (1982) Acta

Haematol. Jpn. 45, 119-128. Sumi,H. , Toki,N. Sasaki,K. , and Mihara,H. (1983) Prog. Fibrinolysis 6,

165-167. Summers ,M. D. and G. E. Smith (1987) A manual of methods for baculovirus

vectors and insect cell culture procedures. Texas Agricultural Experimental Station Bulletin no. 1555, College Station, Tex.

Wun,T. C. , Ossowski,L. , and Reich ,E. (1982) J. Biol. Chem. 257, 7262- 7268.

Wun ,T. C. , Schleuning, W. D. , and Reich ,E. (1982) J. Biol. Chem. 257, 3276-3283.

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