Pergamon .7. Steroid Biochem. Molec. Biol. Vol. 54, No. 5/6, pp. 257-264, 1995

Copyright © 1995 Elsevier Science Ltd 0960-0760(95)00135-2 Printed in Great Britain. All rights reserved

0960-0760/95 $9.50 + 0.00

Direct Expression of Pig Testicular 3 /fl (20fl)-Hydroxysteroid Dehydrogenase in

Escher ich ia coli Shizuo Nakajin,* Takashi Nakajima, Mihoko Uchida, Shuji Ohno

and Masato Shinoda Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41, Shinagawa-ku,

Tokyo 142, Japan

The cDNA coding :for pig tes t icular 3~t//) (20/))-hydroxysteroid dehydrogenase was expressed in Escher ichia coli by placing it under the control of an isopropyl th iogalactos ide (IPTG) inducible tac promote r . P roduc t ion of 3~t/~g (20fl)-HSD was d e m o n s t r a t e d by Western blot t ing and by catalyt ic act ivi ty with 5Gt-di]hydrotestosterone as a subs t ra te for 3~t/Ig-HSD, and proges terone and 17~t- hydroxyproges t e rone as subs t ra tes for 20/)-HSD in the presence of NADPH. The 3~t//) (20/))-HSD enzyme was detec ted in a soluble f rac t ion of the lysate of E. coli added to IPTG to induce the synthesis of the pro'rein. Its molecu la r weight was es t ima ted to be 30.5 kDa by SDS-po lyac ry l amide gel e lectrophoresis (SDS-PAGE). Recombinan t 3at/ig (20i8)-HSD was pur i f ied to appa ren t homogen- eity as d e t e r m i n e d by SDS-PAGE by co lumn c h r o m a t o g r a p h y using DEAE-cellulose. The pur i f ied enzyme reduced not only steroids but also pros tag landins and other carbonyl compounds including aldehydes, ketones and quinones as d e m o n s t r a t e d in nat ive enzymes pur i f ied f r o m pig testes. The amino t e r m i n u s of the pur i f ied enzyme was serine which was coded next to the ATG s ta r t codon, and the sequence of the amino t e r m i n a l 24 residues was ident ical with the coding amino acid in the cDNA; whereas, the amino t e r m i n u s of the native 3~/fl (20fl)-HSD was not detected suggesting tha t the N- t e rmina l amino acid was blocked.

J. Steroid Biochem. ~!olec. Biol., Vol. 54, No. 5/6, pp. 257-264, 1995

INTRODUCTION

We previously repor:ed that a high level of 20fl- hydroxysteroid dehydrogenase (20fl-HSD) activity was present in pig tes~Ees during the neonatal stage [1] and that the enzyme had been purified and character- ized [2-4]. The purified testicular 20fl-HSD catalyzed the conversion of 5~.-dihydrotestosterone (5~-DHT) to both 5~-androstane-3ct, 17fl-diol and 5~-androstane- 3fl,17fl-diol in the presence of NADPH [5]; that is, mammalian testicular 20fl-HSD also had 3~/fl-HSD

*Correspondence to S. Nakajin. Received 19 Dec. 1994; accepted 13 Apr. 1995. Abbreviations: 3~/fl (20fl)-HSD, 3ot/fl (20fl)-hydroxysteroid dehy-

drogenase; PCR, polymerase chain reaction; IPTG, isopropyl-fl- o-thiogalactopyranoside; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- phoresis; 5ct-DHT, 5~-androstan-17fl-ol-3-one; progesterone, 4-pregnene-3,20-dione; 17ct-hydroxyprogesterone, 17ct-hydroxy- 4-pregnen-3-one; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol.

activity for 5e-DHT as demonstrated by prokaryotic 3ct,20fl-HSD from S. hydrogenans [6, 7]. However, the enzymatic properties of testicular 3e/fl (20fl)-HSD were different from the properties of prokaryotic 3ct,20fl-HSD with respect to the specificity of the catalytic reaction and the cofactor requirement [5]. Recently, cDNA encoding 3~t/fl (20fl)-HSD has been isolated from a neonatal pig testis cDNA library and sequenced [8]. Interestingly, the pig 3~/fl (20fl)-HSD has been found to be 85% homologous to human carbonyl reductase [9, 10]. Furthermore, pig testicular 3~/fl (20fl)-HSD exhibits carbonyl reductase-like ac- tivity [8]. In this paper, we describe the direct ex- pression of pig testicular 3~t/fl (20fl)-HSD in Escherichia coli and compare the enzyme activities of the recombinant and the native testicular enzyme. The development of a large scale expression system of this enzyme contributes to the progress of the characteriz- ation of the reaction mechanism of the multifunctional enzyme, 3~/fl (20fl)-HSD.

257

258 Shizuo Nakajin et al.

EXPERIMENTAL

Plasmid construction

The construction of an expression plasmid for 3~/fl (20fl)-HSD from neonatal pig testis was carried out as follows; a cloning vector pBS 52 [8] inserting a full length cDNA which was coded testicular 3~/fl (20fl)-HSD was linearized by Ssp I digestion, and the linearized plasmid was then used as the tem- plate for PCR [11]. The primers used were 5'- G G G A A T T C A T G T C T T C C A A C A C T C G A G - 3 ' as a forward primer for the amino terminal side and 5'- G G G A A T C A G G A C A C A A A T T A T G C A T - 3 ' as a reverse primer for the carboxy terminal side to extend the D N A linker including the EcoRI restric- tion site in the 5' and 3' end of the cDNA. Th e amplified cDNA was digested with the restriction enzyme EcoRI and then purified by agarose gel elec- trophoresis [12]. The expression vector, pKK223-3, was also digested with EcoRI and dephosphorylated with alkaline phosphate. The isolated cDNA was lig- ated with EcoRI cut vector, and the construction was designated pKK223-20fl. The expression plasmid, pKK223-20fl, inserting 3~/fl (20fl)-HSD cDNA was transformed to E. coli JM 105, and the transformants were selected in LB-agar including ampicillin (50 #g/ml). The plasmid with the correct insertion of the cDNA was selected by mapping the digested plas- mid D N A with Bam HI and Xho I. A clone was isolated that included an expression plasmid constructed with the correct insertion. The general techniques for plas- mid construction were performed as described by Sambrook et al. [12].

Expression of recombinant 3~t /fl (20fl)-HSD

The E. coli JM 105 transformed by pKK223-20fl was seeded into LB medium [12] containing 50#g/ml ampicillin and incubated at 37°C until the absorbance at 600 nm reached about 0.2. Expression of 3ct/fl (20fl)- H S D was induced by adding 0.1 m M I P T G . Incu- bation was continued up to a definite time at 37°C with vigorous shaking. After incubation, the E. coli cells were harvested by centrifugation at 300 g for 20 min at 4°C, resuspended with 4 vol. (w/v) of ice-cold 50 m M Tris-HC1 (pH 8.0) that included 1 m M P M S F and 10% sucrose and then treated with lysozyme (1 mg/ml) and 1% Tr i ton X-100. These samples were used for S D S - P A G E and Western blotting. A sonic disinte- grator was used for another preparation method of the bacterial lysate. E. coli cells harvested by centrifugation were washed once with 4 vol. (w/v) of ice-cold 3 m M potassium phosphate buffer (KPB), pH 7.4, including 0 . 1 m M E D T A and 0.1 m M D T T . Th e cells were resuspended with the same buffer and were sonicated with the sonic disintegrator (Taytec VP-30) in an ice bath using 4 m i n pulses with a 10% duty cycle at maximum power, and then they were centrifuged at 105,000 g for 60 min. The supernate obtained was used for the enzyme assay and purification of the enzyme.

Purification of 3ct /fl (20fl)-HSD

After the addition of I P T G (1 mM) for the ex- pression of 3ct/fl (20fl)-HSD, E. coli JM 109 harboring pKK223-20fl in 500ml x 4 LB medium including ampicillin (50 ~tg/ml) was cultured at 37°C overnight with vigorous shaking. The E. coli cells were harvested

Table 1. Substrate specificities of recombinant 3ct /fl (20fl)-HSD purified from E. coli and comparison with the testicular enzyme

Relative velocity Recombinant

Concentrat ion S.A.* Recombinant Test icular Substrate (mM) (nmol /min /mg) (%) (%)

5~ -Dihydrotes tos terone 0.05 55.1 59 90 5fl-Dihydrotestosterone 0.05 35.1 38 29 Testos terone 0.05 8.7 9 0 Progesterone 0.02 3.9 4 10 17~-Hydroxyprogesterone 0.02 15.0 16 10 9 ,10-Phenanthrenequinone 0.01 139 150 362 Menadione 0.25 116 125 63 4-Nitrobenzaldehyde 0.5 92.7 100 100 Phenylglyoxal 1.0 83.6 90 87 Capric aldehyde 0.2 53.3 57 3 Hydr idant in 0.1 120 129 385 Cyclohexanone 10 118 127 96 4-Nitroacetophenone 0.1 71.0 77 20 Camphorqu inone 0.05 226 242 103 Prostaglandin E 1 1.0 15.3 17 0 Prostaglandin E 2 1.0 15.9 17 3

*Specific enzyme activity. Enzyme activity was measured with 44.5 #g of purified enzyme in the presence of 80 / JM

N A D P H in 1.0 ml of 60 m M sodium phosphate buffer (pH 6.5) at 25°C. T h e decrease in absorbance was moni tored at 340 nm. T h e activity with 4-ni t robenzaldehyde was set as 100%, allowing direct comparison with activity of testicular enzyme.

Direct Expression of Pig Testicular 3~/8 (20~)-Hydroxysteroid Dehydrogenase in E. coli 259

by cen t r i fuga t ion and sonicated wi th the sonic d is in te-

grator. T h e superna te ob ta ined by the cen t r i fuga t ion at 105 ,000g for 6 0 m i n was dialyzed in 3 m M K P B

(pH 7.4) i n c l u d i n g 0.1 m M E D T A and 0.1 m M D T T . T h e dialyzed sample was t hen appl ied to a D E A E - cellulose (DE-52) c o l u m n (2.6 x 3 8 c m ) which had been equ i l ib ra ted witlh 3 m M K P B (pH 7.4)-0 .1 m M E D T A - 0 . 1 m M D T T . E lu t i on was carried out wi th a

l inear concen t ra t ion grad ien t ob ta ined f rom 3-100 m M K P B (pH 7.4) i nc l ud i ng 0.1 m M E D T A and 0.1 m M

D T T (750 ml each). Al iquots were assayed for enzyme

activities wi th 5 ~ - D H T as a substrate . As a control exper iment , the superna te for sonicated E. coli cells ha rbor ing only a p lasmid p K K - 2 2 3 was appl ied to the D E A E - c e l l u l o s e c o l u m n in a m a n n e r s imilar to that above.

rime Course

94kl) a-

67kr,~a-

SDS-PAGE

st a b c d e f g h i j st

43kDa.

30kl) a

20. 1 kl)a

14. 4kl) a

W e s t e r n Blotting

Fig. 1. Detection of the expressed 3~c/~ (20~)-HSD enzyme protein in E. coli lysate by SDS-PAGE and Western blotting. The uplper panel is an SDS-PAGE stained with Coomassie blue, and the lower one is a Western blotting, st, puril~ed 30c/~ (20~)-HSD from pig testes. Lane a, lysate prepared from bacteria before addition of IPTG (A660 ~ 0.2); lanes b-j, lysate prepared from bacteria at 0.5, 0.8, 1.1, 1.4, 1.7, 2.1, 2.4 and 3.0 h after the addition of IPTG, as an inducer. For SDS-PAGE, all bacterial samples were suspended with 4 vol. (w/v) of 50 raM Tris-HCl (pH 8.0), including 1 mM PMSF and 10% sucrose, and were treated with lysozyme (1 mg/ml) and 1% Triton X-100. The Western blotting was carried out in 0.1 M Tris-0.192M glycine-0.02% SDS containing 20% methanol from SDS-PAGE gel to a nitrocellulose membrane (Hybond C). For immunostain- ing, pig 3~/~ (20~)-HSD IgG (rabbit) and anti-IgG (donkey) conjugated with horseradish peroxidase were used as the pr imary and secondary antibodies, respectively. The color reaction was carried out with 0.4 mg/ml

diaminobenzidine-0.009% H202 as the substrate of peroxidase.

260

F t -

(9

O U~

u}

A

2 nd s o l v e n t

Shizuo Nakajin et al.

B

e "

(9

m

0

Fig. 2(A,B)--legend opposite.

Vector only

Vector with Insert

2 nd s o l v e n t =

Pig testicular 3~/fl (20fl)-HSD was purified from testes obtained from a neonatal pig after castration according to a previously reported method [2].

E n z y m e assay

An enzyme assay for 3~/ f l -HSD and 20f l -HSD was carried out according to a previous report using radio- active substrates, 5 ~ - D H T , progesterone and 17~- hydroxyprogesterone, which were labeled by 14C at the C4 [1]. Substrate specificity was examined under conditions described previously [8]. Reaction mix- tures consisted of 60 m M sodium p.hosphate (pH 6.5), 44.5 #g of purified 3~/fl (20fl)-HSD, 80 # M N A D P H , and various amounts of substrates (dissolved in 10 pl

of ethyl alcohol) as indicated in Table 1 and were incubated in quartz cuvette (1 cm path length) in a total volume of 1 ml at 25°C. Th e assay of 3~/fl (20fl)- H S D activity for substrate specificity was spectropho- tometrically carried out by monitoring the change in absorbance at 340 nm with time. Furthermore, gas-liquid chromatography was used as previously described to identify the product, the 3~- and/or 3fl-hydroxy steroid corresponding to 5 ~ - D H T [5].

Amino acid sequence

The amino terminal amino acid sequences of purified 3~/fl (20fl)-HSDs were determined by Edman degra- dation using a gas-phase protein sequencer (Applied Biosystems, model 477A).

Direct Expression of Pig Testicular 3ot/fl (20fl)-Hydroxysteroid Dehydrogenase in E. coli 261

C

t -

>

0

2 nd solvent :Fig. 2(C).

Fig. 2. Detection of 20p-HSD (reductase) activity and 3~/~- HSD activity (reductase) of expressed protein in E. coll. Radioautograms obtained by two-dimensional TLC of radio- active metabolites from progesterone (A), 17~-hydroxy- progesterone (B) and 5~-DHT (C) incubated with bacterial lysate in the presence of NADPH. Development of the two- dimensional TLC was carr ied out with benzene :acetone (8 :2 v/v) as a first solvent, and ethyl acetate-n-hexane (7:3 v/v) for panels A and B and benzene-ethyl acetate (2:1 v/v) for panel C as a second solvent. Vector only: incubation with the lysate prepared f rom bacteria t ransformed with the expression vector. Vector with insert: incubation with the lysate prepared f rom bacteria t ransformed with the ex- pression vector inserted into testicular 3~/~ (20p)-HSD cDNA. The assigned spots are (a) progesterone, (b) 20p- hydroxy-4-pregnen-3-one, (c) 17~-hydroxyprogesterone, (d) 17~,2018-dihydroxy-4-pregnen-3-one, (e) 50¢-DHT, (f), 5~-

androstane-3~ (or ~), 17~-diol.

C

e,- o

4-,, o

a

L__.,.,_ I I I I I

0 10 20 30 40

Time (min)

Fig. 3. Gas-liquid chromatography of steroid metabolites of 5~-dihydrotestosterone. A methylene dichloride extract was silylated with TMS-imidazole at room temperature . Peak assignments: (a), 5~-androstane-3~,17p-diol; (b) 5~-an-

drostane-3~,17p-diol; (c) 5~-DHT.

Analytical methods

P o l y a c r y l a m i d e gel e l ec t rophores i s in the p re sence o f s o d i u m dodecy l sulfate ( S D S - P A G E ) was ca r r i ed out b y the m e t h o d o f L a e m m l i [13] u s ing 10% a c r y l a m i d e gels. T h e m o l e c u l a r we igh t ma rke r s used for S D S - P A G E were p h o s p h o r y l a s e b (94 kDa) , bov ine s e r u m a l b u m i n (67 kDa) , o v a l b u m i n (43 kDa) , ca rbon ic a n h y - drase (30 kDa) , soybean t r yps in i nh ib i t o r (20.1 kDa) and c t - l ac ta lbumin (14.4 kDa) .

T h e p r o t e i n concen t r a t ions were e s t ima ted by the m e t h o d o f L o w r y et al. [14] us ing c rys ta l l ine bov ine s e r u m a l b u m i n ( A r m o u r P h a r m a c e u t i c a l Co. , F r a c t i o n V) as a s t anda rd . D u r i n g the c o l u m n c h r o m a t o g r a p h y , the concen t r a t i on o f the p r o t e i n was e s t ima ted by m e a s u r i n g the a bso rba nc e at 280 nm.

W e s t e r n b lo t t i ng was ca r r i ed ou t as p r ev ious ly d e s c r i b e d [15].

Chemicals

P l a s m i d vec tor , p K K 2 2 3 - 3 , and E. coli J M 105 were o b t a i n e d f rom P h a r m a c i a P - L Biochemica l s Inc . (Mi lwaukee , W I ) .

Res t r i c t i on enzymes , EcoRI, SspI, B a m H I and Xh o I , alkal ine p h o s p h a t a s e (calf in tes t ine) and a l ig- a t ion ki t were p u r c h a s e d f rom T a k a r a Shuzo Co. L t d .

262 Shizuo Nakajin et al.

( Kyo to , Japan) . A m p l i T a q D N A p o l y m e r a s e was f rom P e r k i n - E l m e r Cetus I n s t r u m e n t (Norwa lk , CT) . Var i - ous add i t i ona l ma te r i a l s and chemica l s were ob t a ined f rom sources p r ev ious ly r e p o r t e d [1 ,2 , 5 ,8] . O t h e r r e a g e n t s were o f the bes t g rade avai lable and were ob t a ined f rom W a k o Chemica l s (Tokyo , Japan) .

R E S U L T S AND D I S C U S S I O N

Construction of the expression plasmid

T o p r e p a r e 3~lfl ( 2 0 f l ) - H S D c D N A for inse r t ion into the expres s ion p l a smid , the P C R t echn ique was used . Ampl i f i ca t ion of the 3ct/fl ( 2 0 f l ) - H S D sequence ,

wh ich was i n c o r p o r a t e d in the c lon ing vec to r pBS52 , was p e r f o r m e d by P C R . T h e fo rw a rd and reverse p r i m e r s were des igned to create E c o R I r e s t r i c t ion sites at the 5 ' - s i t e of the A T G star t codon and at the 3 ' - s i t e o f the 10 nuc leo t ides d o w n s t r e a m f rom the T A A s top codon. T h e ampl i f i ed c D N A which was pur i f ied by agarose gel e l ec t rophores i s , was l iga ted wi th the ex- p re s s ion p l a s m i d p K K 2 2 3 - 3 , wh ich had a d e p h o s p h o - ry la ted E c o R I cut, and the cons t ruc t ed p l a smid p K K 2 2 3 - 2 0 f l was t r a n s f o r m e d to E. coli J M 105. A clone tha t i n c l u d e d an express ion p l a s m i d c o n s t r u c t e d wi th the cor rec t inse r t ion was i so la ted , and the ex- p re s s ion of the p ro t e in was p laced u n d e r the con t ro l o f

I P T G .

(A) 1.5

E 1.0

O ¢O

® I3 ~ 0.5

O fn

.Q < [

0

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i i Ii / " I i! tl -" ~ "1 ~s°

II / i t / i~ I I / ! k / ':i !i , . / ...' \ -I ,00 i i ! ! w .# t / ' ~ ..., .,. /

• ,. I 1 # i I ."-, " " i • ... I I I " ",-. ! '.....! 4 s o • , ll,.-' / v /

i ~. ~ ] . . .~ i l ~ . . _ . , = . ~ : ~ . = : 3 J i " t . . . . . . • /

2 0 4 0 6 0 8 0 1 0 0 F rac t ion No. (15 ml / f rac t lon )

50 I

4° I

" • 3 0 .~

e--

. . 2 0 c u O (3 ®

0 I -

Q .

(B) 1.51- .:. /

/ i i / . i / ~! / . ; ; f ,

_ 1 0 ! | / !~ • / ' : / .t%1 ' " / i ~ o ~ / i l . , , , ,

• : . ! ® ! : - / i . ; Xq / L,".- '""

I I / i t s - x ,,. i "-,. ,.-"X /

e l \ .., , ,,..,. ,,./

0 2 0 4 0 6 0 8 0 1 0 0 F rac t ion No. (15 rn l / f ract lon)

- 5 0

2 ( 0 - 40

153 • - 30

10:) - 2 0

5( I • 10

0 . 0

I I E c ._o

t -

8 c O

o ®

8 e -

Fig . 4. DEAE-cellulose column chromatography of E. coli lysate. Supernatant fluids prepared from the lysate of IPTG induced E. coli t ransformed with the expression vector inserting cDNA (A) and the expression v e c t o r only (B) were dialyzed with 3 M KPB (pH 7.4) including EDTA and DTT (0.I mM each). Dialyzed samples (1.1 g) were applied to a DE-52 column (2.6 × 38 cm) w h i c h h a d been equil ibrated with a dialysis buffer. The column was then eluted with a l inear concentration gradient of KPB from 3 to 100 raM, and 15 ml of t h e f r a c t i o n w a s collected. The proteins were monitored by measuring the absorption at 280 nm, and aliquots were

assayed for enzyme activity.

Direct Expression of Pig Testicular 3~//~ (20~)-Hydroxysteroid Dehydrogenase in E. coli

SDS-PAGE Western Blotting

263

94K-

67K-

43K-

30K-

20.1K-

14.4K-

M a b c d

Fig. 5. SDS-PAGE and Western blotting of the recombinant 3~,/p (20p)-HSD purified from E. coli lysate. Lane M, molecular weight markers; lane a, native 3~/p (20~)-HSD purified from pig testieular eytosol; lanes b and c, the lysate of IPI 'G induced E. coli transformed with the expression vector only and the expression vector

inserting eDNA, respectively. Lane d, recombinant 3~/p (20p)-HSD purified E. coli lysate.

Expression of 3ot/fl (20fl)-HSD enzyme

T h e expression product was detected by Coomassie blue stain and Western blot after S D S - P A G E as shown in Fig. 1. T h e expression product in the lysate of the bacteria had migrated to almost the same position as the native 3~/[3 (20[3)-HSD and increased with the passage of time, after the induction of I P T G ; whereas, the bacteria which was not induced by I P T G did not produce an expression product. In addition, the ex- pression product was localized in the supernatant fluid f rom the bacteria lysate and was not detected in the 105,000 g precipitate.

Recombinant 3~/[3 C20[3)-HSD expressed in E. coli was catalytically active with some steroids in the pres- ence of N A D P H . As shown in Fig. 2(A and B), when a soluble fraction f rom the bacterial lysate was incu- bated with progesterone or 17~-hydroxyprogesterone in the presence of an NADP H-gene ra t i ng system, 20[3-hydroxy-4-pregnen-3-one or 17~,20[3-dihydroxy- 4-pregnen-3-one was produced, respectively. In ad- dition, as shown in Fig. 2(C), when 5~-DI- IT was incubated as a substrate instead of C21 steroids, 5~- androstane-3~ (or [3), 17fl-diol was produced by catal- ysis of the recombinant enzyme. Fur thermore , based

on the analysis of gas-l iquid chromatography of the product after incubation using the purified recombi- nant enzyme, as shown later, both the 5~-androstane- 3~,17fl-diol and 5~-androstane-3fl,17fl-diol were detected as the products f rom 5 ~ - D H T (Fig. 3). Ac- cordingly, the recombinant 3~/[3 (20f l ) -HSD expressed in E. coli had 3~/[3-HSD activity against 5 ~ - D H T (C19 steroid) and 20f l -HSD activity against progesterone and 17~-hydroxyprogesterone (C21 steroids).

Purification of recombinant 3o~/fl (20fl)-HSD

T h e recombinant 3~/[3 (20[3)-HSD expressed in E. coli J M 105 was purified f rom the cell lysate using

Table 2. Amino acid sequences of the amino terminal region of testicular and recombinant 3~/[J (20fl)-HSD

1 10 20

Deduced from cDNA MSSNTRVALVTGANKGIGFAIVRDL Recombinant enzyme S SNTRVALVTGANKGI GFAIVRDL Native enzyme ND

The numbers above the sequence indicate the residue number from the animo terminus.

ND, Amino terminus was not detected by Edam degradation.

264 Shizuo Nakajin et al.

D E A E - c e l l u l o s e (DE-52 ) c o l u m n c h r o m a t o g r a p h y (Fig . 4). T h e pur i f i ed enzyme showed a s ingle b a n d on S D S - P A G E and W e s t e r n b lo t t i ng as shown in Fig . 5, w i th the same mig ra t i on as tha t o f the nat ive 3~/fl

( 2 0 f l ) - H S D pur i f i ed f rom the tes t i cu la r cy tosol o f a neona ta l pig. T h e mo lecu l a r weight was e s t ima ted to be 30.5 k D a by S D S - P A G E . T h e y ie ld o f the r e c o m b i - nan t enzyme f rom the bac te r ia l cu l tu re was 20 mg/1 of m e d i u m and was 3 .5% f rom the bac te r ia l lysate.

the t r a n s f o r m e d E. coli cell lysates us ing D E A E - cel lulose (D E -52 ) c o l u m n c h r o m a t o g r a p h y wi th a y ie ld of 20 mg/1 o f cu l ture . T h e pur i f i ed r e c o m b i n a n t en- zyme showed enzyme act iv i t ies 3 ~ / f l - H S D ( reduc tase) and 2 0 f l - H S D ( reductase) , and ca rbony l r educ ta se - l ike ac t iv i ty in the p resence o f N A D P H , as shown in the na t ive enzyme. H o w e v e r , the a m i n o t e r m i n u s o f the r e c o m b i n a n t enzyme was d i s t inc t f rom tha t o f the na t ive enzyme.

Amino terminal sequence o f 3ct/fl ( 2 0 f l ) - H S D

T h e a m i n o t e r m i n a l sequence o f 3~t/fl ( 2 0 f l ) - H S D is shown in T a b l e 2. T h e a m i n o t e r m i n u s of the r e c o m b i - nan t enzyme was ser ine and the sequence o f the 20 a m i n o t e rmina l res idues was ident ica l to tha t d e d u c e d f rom c D N A . Based on these data , it can be d e m o n - s t r a t ed tha t af ter t r ans l a t ion of the A T G star t codon to fo rmy l m e t h i o n i n e , its m e t h i o n i n e was r e m o v e d by m e t h i o n i n e amino pep t ida se f rom E. coli, as p rev ious ly r e p o r t e d by Ben-Bassa t et al. [16]. On the o the r hand , the a m i n o t e r m i n u s o f na t ive 3~/fl ( 2 0 f l ) - H S D was no t de t ec t ed by E d m a n d e g r a d a t i o n sugges t ing tha t the amino t e r m i n u s of the enzyme was b locked as has been r e p o r t e d for ca rbony l r educ tase [9].

E n z y m e act iv i ty o f the recombinant 3~/fl ( 2 0 f l ) - H S D

T h e specif ic i ty o f r e c o m b i n a n t 3a/f l ( 2 0 f l ) - H S D for the subs t ra te , i nc lud ing some s tero ids and o the r car- bony l c o m p o u n d s , was e x a m i n e d us ing the c o m p o u n d s l i s ted in T a b l e 2 for c o m p a r i s o n wi th the nat ive en- zyme , wh ich exhib i t s ca rbony l r educ ta se - l ike ac t iv i ty [8]. T h e pur i f ied r e c o m b i n a n t enzyme r e d u c e d 5ct- D H T at a compa ra t i ve ly h igh rate in the p resence of N A D P H . Iden t i f i ca t ion of the p r o d u c t was p e r f o r m e d b y g a s - l i q u i d c h r o m a t o g r a p h y . As shown in Fig . 3, t r ime thy l s i l y l ( T M S ) - d e r i v a t i v e s of 5 ~ - a n d r o s t a n e - 3~,17f l -d io l and 5~t -andros tane-3f l ,17f l -d io l were de - t ec ted as the p roduc t . F u r t h e r m o r e , the r e c o m b i n a n t enzyme r e d u c e d no t on ly s te ro ids bu t also p ros t ag l an - d ins and o the r ca rbony l c o m p o u n d s , i nc lud ing a lde- hydes , ke tones and qu inones . T h e s e ca rbony l c o m p o u n d s were r e d u c e d m u c h m o r e eff iciently than the s te ro ids and p ros t ag land ins . T h e rate of r e d u c t i o n o f 4 - n i t r o b e n z a l d e h y d e was set at 100%, a l lowing d i rec t c o m p a r i s o n of the resul ts w i th na t ive 3~/fl ( 2 0 f l ) - H S D iso la ted f rom neona ta l p ig testes [8]. I t was f o u n d tha t the subs t r a t e specif ic i ty o f the r e c o m b i n a n t e n z y m e was s imi la r to tha t o f the na t ive enzyme. H o w e v e r , a s l ight d i f ference was o b s e r v e d which m a y be a t t r i b u t a b l e to the d i f ference in the a m i n o t e r m i n u s reg ion be tween the na t ive and r e c o m b i n a n t enzymes .

I n conc lus ion , a p l a s m i d for d i rec t express ion in E. coli of func t iona l p ig t e s t i cu la r 3~/fl ( 2 0 f l ) - H S D was cons t ruc t ed f rom the fu l l - s ize c D N A for the enzyme. T h e r e c o m b i n a n t 3~/fl ( 2 0 f l ) - H S D was pur i f i ed f rom

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