Gene, 118 (I 992) 239-245 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

239

0378-1119/92/$05.00

06608

Production

of mature bovine pancreatic

(PCR amplification;

Antonio

periplasm;

Tarragona-Fiol,

RNase;

signal sequence;

Christopher

(Received

by J.K.C.

Knowles:

29 January

translocation;

J. Taylorson,

London Biotrchnolqg~ Ltd., Department of Biochemistry

ribonuclease two-cistron

in Escherichia

coli

vector)

John M. Ward and Brian R. Rabin

and Molecular Biology, IJnivrrsitJ College London. London. UK

1992; Revised,!Acccpted:

10 March:13

March

1992; Received

at publishers:

18 May 1992)

SUMMARY

The coding sequence for the bovine pancreatic ribonuclease (RNase) precursor has been cloned and produced in Escherichiu coli using the polymerase chain reaction (PCR) technique. A PCR amplification has been carried out utilizing as template the recombinant plasmid, pQR138, which contains the coding sequence for the RNase precursor, and primers that allow for the addition of new sequences at the 5’ and 3’ ends of the coding sequence. The resultant fragment contains two coding sequences, one for a hexapeptide and the other for pre-RNase. This fragment has been cloned into the expression vector, pKK223.3, under the control of the tat promoter, to form a two-cistron vector. Upon induction with IPTG, E. coli cells harboring this construct generate a bicistronic mRNA which upon translation produces a hexapeptide and pre-RNase. The RNase precursor is efficiently translocated into the periplasmic space of E. coli. Upon translocation, the signal sequence is removed generating mature RNase. Formation of the disulfide bridges in RNase is facilitated by the oxidative environment of the periplasm and a fully active protein is obtained. RNase produced in E. coli has been purified to homogeneity by cation-exchange chromatography, and the removal of the signal sequence has been verified by N-terminal sequencing. The total process from inoculation of media to obtaining pure and fully active recombinant RNase is achieved in 48 h.

INTRODUCTION

Bovine pancreatic RNase A has been one of the most comprehensively studied enzymes in the past two decades. Its crystal structure has been elucidated (Borkakoti et al., 1982) and its physico-chemical properties are well characterized (Eftink and Billonen, 1987). The active site structure (Llorens et al., 1989) and the mechanism of catalysis are well understood (Findlay et al., 1962). However, the

Correspondence to: Mr. A. Tarragona-Fiol, and Molecular WClE

Biology, University

Department

College London,

of Biochemistry

Gower

St., London

6BT, UK. Tel. (44-71) 387 7050, ext. 2174; Fax (44-71) 387 7193.

Abbreviations:

A, absorbance

(1 cm); aa, amino acid(s);

Ap, ampicillin;

specificity that the enzyme shows towards different dinucleotide substrates is less well understood. Available data indicate that the precise nature of the base component of the leaving group of the substrate has a major inlluence on the catalytic rate as measured by k,,,. Thus, in a series of dinucleotides, CpA, CpG, CpC and CPU, the measured values of k,,, decrease from 3000/s to 27/s with only very small changes in K, (Witzel and Barnard, 1962). To gain a real insight into the interactions between the enzyme and

MES, tergent);

2-[N-morpholino]ethanesulfonic nt, nucleotide(s);

acid: NP-40,

ORF, open reading

Nonidet

P-40 (de-

frame; ori, origin of repli-

cation; PAGE. polyacrylamide-gel clectrophoresis; PCR, polymerase chain reaction; PolIk, Klenow (large) fragment of Escherichia co/i DNA polymerase

I; pre-RNase,

gene encoding

for the precursor

form of RNase;

bp, base pair(s); CpA, cytidylyl-3’:5’-adenosine; CpC, cytidylyl-3’:5’cytidine; CpG, cytidylyl-3’:5’-guanosine; CpU,cytidylyl-3’:S’uridine:

R, resistance/resistant; RBS, ribosome-binding site(s); RNase, ribonuclcase; RNase, gene encoding RNasc; SDS, sodium dodecyl sulfate; Tc,

IPTG, isopropyl-P-D-galactopyranoside;

tetracycline;

catalytic second):

kb, kilobase

or 1000 bp: k,,,,

constant (moles of substrate converted per mol of enzyme per k’,,,, Michaelis rate constant; MCS, multiple cloning site(s);

Tet/Z, a thermostable

UV, ultraviolet,

polymerase

[ 1,denotes plasmid-carrier

from Thermus rhermophilus; state.

240

A

4 - Ligation into pUC 18 pKK223.3 digested with Eco RI and dephosphorylated L?coRl WCs) lac I i -

Digestion with EcoRI - Electrophoresis - Gel extraction of small fragment.

B Met ------

Phe

Leu

Glu

Asp

Asp

r’ preRNa.5=

Primer 10: S- GCGC GAATTC ATG TTC TTG GAG GAT GAT TGATG GCT CTG AAG TCC C -3’ EcoRI

RBS

z=k stop codon

Primer 11: S- TGTC GAATTC GGCCTTAGGTAGAGACCTAC vector and sequence

-3’

stop codon

EcoRl Fig. I. Construction of a two-cistron amplification using primers 10 and

start - codon

of primers

used in PCR. (A) New constructs. Plasmid pQR138 serves as template for a PCR two-cistron fragment, section b) thus generated, was treated with PolIk (10 units)

II. The PCR fragment (designated

and all four dNTPs for 30 min at 37°C to repair ragged ends. EcoRI (20units) was used to cleave the DNA for 3 h at 37°C prior to ligation into prcviously digested and dephosphorylated pUC18. Ligation was carried out overnight at room temperature. Transformation of ligated products was performed as described

by Maniatis

DNA sequencing

et al. (1982). Plasmid

(Fig. 2). The plasmid

DNA was isolated

containing

the two-cistron

from the recombinant fragment

colonies

was designated

and the sequence

pQRl62.

This plasmid

was verified using double-stranded was digested

with EcoRI

and the

products of the digestion were separated by agarosc gel electrophoresis. The two-cistron fragment was extracted from the gel and ligated overnight at room temperature into digested and dephosphorylated pKK223.3. After transformation and plasmid isolation, the restriction enzyme Pstl was used to ascertain orientation with respect to the tat promoter. The plasmid chimaera Restriction enzyme digestions and dephosphorylations were performed

having the two-cistron fragment in the right orientation was designated pQRl63. as described in Maniatis et al. (1982). (B) Sequence of the primers used for the

PCR to generate the two-cistron fragment (sections b and c). The primers wcrc synthesized using cyanoethyl phosphoramidites thesizcr (Milligcn/Milliporc) and purified by a combination of anion exchange and reverse-phase chromatography.

in a Cyclone

DNA syn-

241 cistron vector, the latter being facilitated by the net positive charge of the protein at a pH near neutrality. The

the dinucleotide substrates, site-directed mutagenesis studies are needed and this requires a system which will enable the rapid expression and isolation of RNase. Recombinant DNA techniques have been used to design an expression system which enables the production of mature RNase and mutants thereof (A.T-F., C.J.T., J.M.W. and B.R.R., unpublished) in the periplasmic space of E. coli. A number of factors affect the production of eukaryotic proteins in prokaryotes, these include degradation of the protein (Shen et al., 1984), formation of inclusion bodies due to the protein insolubility (McGeehan and Benner, 1989) and instability of mRNA (Newbury et al., 1987). Another problem encountered is that of secondary struc-

purified recombinant RNase has been shown to have an activity equivalent to that of commercial RNase.

RESULTS

stop

Stall

codon

codon 1

was used as a plasmid host and for expression studies. The plasmids used were: pQR138 (kindly provided by Prof. A. Furia; Carsana et al.,1988), a plasmid chimaera comprising pBR322 and a 1.14-kb fragment containing a 450-bp cDNA fragment which codes for bovine pancreatic RNase precursor; pUC18 and pKK223.3 (PharmaciaLKB) (Fig. 1A). Small- and large-scale plasmid isolation was performed as described by Birnboim and Doly (1979). Growth under standard conditions was carried out in either nutrient broth (Oxoid) or nutrient agar (Oxoid nutrient broth solidified with 2% (w/v) Bacto agar). For expression studies, a medium was used which contained (per ml): KH,PO, (2.3 mg)/K,HPO, (3.78 mg)/Bacto tryptone (12 mg)/yeast extract (24 mg)/0.4% (v/v) of glycerol (Tartoff and Hobbs, 1987). Growth of cells producing RNase was carried out at 28°C. Transformation of competent cells with plasmid DNA was carried out as described in Maniatis et al. (1982).

? codon RBS I_= GAA~C~‘ITCTTGGAGGATGATTGATGGCTCTGATC Met F%e Leu Glu Asp Asp Met Ala LXI Lys EmFu \_“,/

DISCUSSION

(a) Bacteria, plasmids and media The E. coli strain JM107 {endA 1, g-v-96, thi-1, hsdR17, supE44, relA1, d(fac-proAB); Yanisch-Perron et al., 1985)

ture of the mRNA (Hall et al., 1982). Preliminary studies using Beckman Microgenie software to simulate the secondary structure of the mRNA that would be produced following ligation of RNase precursor-coding sequence into pKK223.3, indicated that efficient expression would not be possible due to the formation of a stem-loop containing the RBS and the start codon of pre-RNase (data not shown). Available data indicated that the construction of a twocistron vector could solve the problem (Schoner et al., 1984; Mashko et al., 1990). Therefore, a small peptide (first cistron) was attached 5’ to the N terminus of pre-RNase (second cistron) using the PCR technique. Computer analysis shows that this construct minimizes the secondary structure of the mRNA. The aims of the present study were the production and purification of recombinant RNase in E. coli using the two-

Start

AND

Ser

Leu Vd

La

La

Ser

La

Lea Vd

-___-) ii71rea el¶sraffom

secomd

CB~%lpOrm

r+

RNase

CTGGTGCTGCTGCTGGTGCGGGTCCAGCCTTCCCTGGGCA L~JJ W LRJ L.eu h Val & Val Gin Pro Ser Len Gly GAGCGGCAGCACATGGACTCCAGCACTTCCGCTGCCAGCAG Glu Arg Gin His Met Asp .%I Ser llu Ser Ala Ala Ser

Lys

Glu

llw

Ala

Ala

Ala

Ser

Ser Am

Tyr

Cys Am

Lys

Phe

Gin Met

ATGAAGAGCCGGAACCTGACCAAAGATCGATCGATGC~GCCAGTG~CACC~GTGCACGAGTCC

Met Lys

Se

Arg

Au,

Leu

7lu

Lys

Asp

Arg

Cys

Lys

Pm

VaJ

Asn

7h

Phe

Val

His

Glu

Ser

llu

Asn

CTGGCTGATGTCCAGGCCGTGTGCTCCCAG~TG~GCCTGC~G~T~CAGACC~T Leu Ala Asp Val Gin Ala Vd Cys Ser Cl,, Lys Asn Val Ala Cys Lys

Asn

Gly

Gin

TGCTACCAGAGCTACTCCACCATGAGCATCACCGACTGCCC CYs TY Gin Ser Tyr Ser Eir Met Ser ile llu Asp Cys Ae

Gly

Ser

SET Lys

Glu

7hr

CCCAACTGTGCCTACAAGACCACCCAGGCGAATAAACACACATCA~GT~C~GTGAG~~C PIW A.m CYS Ala Tyr Lys ‘I& llu Gin Ala Asn Lys His ne lie Vd Ala Cys

Glu

Gly

Ty

Asn

CCGTACGTGCCAGTCCACATGCTTCAGTGTAGTGTAGGTCTCTACCTAAGGCCGAATTC Pro

Tyr

Val

Pm

Vd

His Phe Asp Ala

SW Vd

StoD

E&RI

cod& Fig. 2. Sequence

of the two-cistron

fragment

obtained

by double-stranded

DNA sequencing

corresponds to a hexapeptidc (first cistron) and to pre-RNase (second cistron). et al. (1977) using a commercial T7 Sequencing kit (Pharmacia-LKB).

Double-stranded

using pQR162

as template

DNA sequencing

(Fig. IA). The aa sequence

shown

was carried out by the method of Sanger

242 (b) Design of primers for PCR Primers complementary to the 5’ and 3’ ends of the coding sequence for RNase precursor were synthesized and used for a PCR amplification. Primer 10 contains additional sequence information which will incorporate a short ORF in front of the RNase precursor and an EcoRI restriction site with four additional nt to ensure correct cleavage (Fig. 1B). Primer 11 contains the termination codon for RNase, an EcoRI site and an additional 4 nt to ensure correct cleavage (Fig. 1B). The two-cistron fragment resulting from PCR amplifica-

tion using primers 10 and 11, contains two sets of coding sequences: one for a hexapeptide and the other for RNase precursor. Transcription of this fragment produces bicistronic mRNA which upon translation generates a hexapeptide and the pre-RNase. (c) Production of a two-cistron The PCR reaction contained

fragment using PCR pQR138 as template (100

ng)/primers 10 and 11 (50 pmole/each)/all four dNTPs (0.2 mM each)/20 mM Tris.HCl pH S.O/lS mM (NH,),S04/2 mM Mg,Cl/O.OS% NP40/0.05% Tween 20, in a total vol-

lb-

1.4-

12-

I

j

IO-

I r--

06-

1 i-

0.4 -

I \

\ I

02-

--

1 / I

!

I_Ll-

8C

Fig. 3. Purification extract

560

of recombinant

(from a 5-l culture)

dimensions:

RNase.

720

(A) Isolation

was filtered through

of the posttively charged

proteins from the periplasmic

extract by isocratic

a cellulose nitrate filter (pore size, 0.45 pm) and loaded onto a column

25 cm x 16 mm) at a flow rate of 4 ml/min. The column was then washed

containing

elution. The periplasmic S-sepharose

with 50 mM MES pH 6.5 (250 ml) followed by an isocratic

(gel bed elution

with SO mM MES pH 6.511 M NaCl (250 ml) to elute bound proteins. The fractions corresponding to the peak obtained after isocratic elution containing positively charged proteins were pooled in a ultrafiltration cell (Amicon, model 8050). Filtration of the sample was carried out through a YM-10 ultrafiltration membrane (M,. cutoff > 10000). The retentatc was desalted on a G-25 column (NAP-25, Pharmacia-LKB). (B) Purification of recombinant RNase from the desalted pool of positively charged proteins by FPLC (fast protein liquid chromatography) using a Mono-S column (Pharmacia-LKB). Elution was carried out using buffer M (50 mM MES pH 6.5) and buffer N (50 mM Mes pH 6.5/l M NaCI). A gradient was applied from 0 to 350 mM NaCl in 20 min at a flow rate of 1 ml/mitt. The peak obtained at 160 mM NaCl showed RNase activity (arrow). Recombinant RNase was then desalted on a G-25 column (NAP-5, Pharmacia-LKB). (C) Re-run of the fraction elutcd at 160 mM NaCl in panel B; the major peak is recombinant RNase (arrow). The NaCl gradient

is represcntcd

by thin dashed

lines.

243 ume of 50 or 100 ~1. The reaction components were rapidly mixed followed by centrifugation at 10000 x g (average) for 2 s at room temperature. An equal volume of paraffin oil was added to avoid evaporation during the reaction. Template DNA was fully denatured by incubation for 5 min at 92 oC and the reaction was initiated by the addition of Tet/Z polymerase (2 units, 1 unit/pi). The reaction was carried out in a thermal cycler (Techne 2000) for 25 cycles of denaturation at 92’ C for 1.5 min, annealing at 55 “C for 1 min, and extension at 72” C for 1.5 min. (d) Synthesis and purification of recombinant coli [pQR163] cells

RNase in E.

The plasmid pQR163 contains two RBS, one provided by the tuc promoter of the vector and the other, for translation of the second cistron, is contained within the coding sequence of the first cistron. The mRNA produced upon IPTG induction of E. co/i cells harboring pQR163 is bicistronic and starts from the tat promoter. The first cistron encodes a 6-aa peptide (Met-Phe-Leu-Glu-Asp-Asp). The stop codon of the first cistron and the start codon of the second cistron overlap such that ribosomes will continue translation of the mRNA and produce pre-RNase (Fig. 2). The synthesized precursor form of RNase is translocated

to the periplasm and N-terminal sequencing has shown that the signal sequence is correctly cleaved. The oxidative environment of the periplasm allows the correct folding of RNase to form the native enzyme as evidenced by the recovery of fully active enzyme (Figs. 5 and 6). (1) Induction of E. coli[pQR163/

cells with IPTG

E. coli [pQR163]

cells were grown in 5 liter of media containing 100 pg Ap/ml for 4-5 h at 28°C. An aliquot (1 ml) was removed and growth was assessed by measuring the absorbance (A) at 550 nm. When cells were in the exponential phase of growth IPTG was added to a final concentration of 0.5 mM and growth of cells was continued overnight at 28°C with shaking. The A,,, nm at the time of harvesting was 7.

Fig. 5. Recombinant containing

2%(w/v)

RNase

activity

on RNA

agar (Bacto-agar)/lOO

agar

plates.

A solution

mM MES pH 6.5/0.3%

(w/v)

yeast RNA was autoclaved and poured in Petri dishes. Round holes were made in the RNA agar using a sterile cork-borer and recombinant or Fig. 4. Assessment 0.1% SDS-16”/ mercial

RNase

of purity of recombinant polyacrylamide

(M, 13700).

RNase

on a silver-stained

gel. Lanes 1 and 7 correspond

Lanes 3-5,

contain

positively

to com-

charged

pro-

commercial RNase (0.1-25 pg) was applied in a volume of 150 ~1. The plates were incubated for 2-4 h at 37°C. To visualize RNase activity, 2 M HCI was poured

over the plate. A clear zone is produced

hole were active RNase has been introduced

around

the

and RNA that had not been

teins obtained after isocratic elution of the periplasmic extract from E. coli[pQR163] cultures and induced with different concentrations of IPTG

hydrolyzed forms a cloudy precipitate. The top plate shows the halos produced by different amounts of commercial RNase: A, 100 ng; B, 1 pg;

(0.5, 2 and 0 mM respectively).

C, 5 ng; D, 25 pg; E, 10 pg; g, control

Lane 6, as lanes 3-5

but the culture

contained E. cnli [pKK223.3] cells (control). Lane 2, purified recombinant RNase after ion-exchange chromatography (Fig. 3C).

(no enzyme).

The bottom

plate

shows the halos produced by recombinant RNase: X, 114 ng; Y, 570 ng; Z, an aliquot (10 ~1) of the periplasmic extract of E. c&[pQR163] cells.

244 (3) PuriJication of recombinant RNase Cation exchange chromatography was used to obtain all the positively charged proteins from the periplasmic extract (Fig. 3A). Proteins obtained after isocratic elution were separated by electrophoresis on a 16% PAGE-O. 1 y0 SDS (Laemmli, 1970) and silver stained (Fig. 4). Recombinant RNase was purified from the pool of positively charged proteins by cation exchange chromatography (Fig. 3B and C). Assessment of the purity of recombinant RNase by PAGE-SDS electrophoresis and silver staining (Fig. 4) clearly shows that this combination of techniques results in purification of the protein to homogeneity. Chromatographic procedures are achieved in 4 h. RNase activity of the recombinant enzyme was estimated on RNA agar plates (Fig. 5) and on the hydrolysis of CpA (Fig. 6) showing an equal specific activity to that of the commercial enzyme.

A 265

0.06

0.02

t (min) Fig. 6. Hydrolysis

of cytidylyl-3’:5’-adenosine

mercial RNase. The hydrolysis temperature

in l-ml,

by recombinant

l-cm path cuvets (Hellma).

action was 1 ml. Reactions

contained

The volume of the re-

0.1 mM CpA in 0.1 M Tris’acetate

pH 6.5 and were initiated by adding RNase to a final concentration nM (50 ng). The hydrolysis taining

0.1 M Tris.acetate

and com-

of CpA by RNase was carried out at room

of CpA was measured,

against

of 3.65

(e) N-terminal sequencing of recombinant RNase The produced and purified recombinant RNase was sequenced on an Applied Biosystems 470A automated gas phase peptide sequencer using Edman degradation with identification of the phenylthiohydantoin aa derivatives by high pressure liquid chromatography (HPLC). The sequence of the purified enzyme was found to be identical to that of the bovine enzyme (data not shown), indicating correct and efficient removal of the RNase signal sequence during the process of translocation.

a blank con-

pH 6.5 and 0.1 mM CpA, by an increase

in

%hi “lll~

(2) Isolation of the periplasmic proteins The release of the periplasmic proteins was carried out using a modified spheroplast/osmotic shock procedure developed by C. French (C. French, J.M.W. and P. Dunnill, unpublished). Cells from an overnight culture (5 1) were pelleted by centrifugation at 8300 x g (average) for 10 min at 4” C. The cell pellet was resuspended in 60 ml of 200 mM Tris.HCl pH 7.5/20% (w/v) sucrose (RNase free)/1 mM Na,EDTA/lysozyme (500 mg per ml). The suspension was left for 15 min at room temperature. An osmotic shock was obtained by adding an equal volume of sterile water and mixing thoroughly. The mixture was left for 15 min at room temperature. Spheroplasts were pelleted by centrifugation at 100000 x g (average) for 90 min at 10°C. Protein concentration was determined according to the method of Bradford (1976). This spheroplast/osmotic shock method (C. French, J.M.W. and P. Dunnill, unpublished) releases over 900,;, of the periplasmic proteins and in this work no RNase was detectable in either the supematant after a 50-fold concentration, or in the cellular pellet.

(f) Conclusions (I) Several attempts have previously been made to produce RNase in E. coli either as an intracellular inactive hybrid protein linked to b-galactosidase by a tetrapeptide (Nambiar et al., 1987) or as inclusion bodies containing formylated N-terminal Met (McGeehan and Benner, 1989). The enzyme has also been expressed using Bacillus subtilis as a host and in this system the product was secreted into the medium, with associated problems due to protcolysis (Vasantha and Filipula, 1989). (2) The system described here utilizes the eukaryotic signal sequence of RNase precursor to export the protein to the periplasm of E. co/i. During translocation the signal sequence is removed. The site of cleavage is correct since the sequence of the first 20 aa is identical to that of the bovine enzyme. Due to the oxidative environment of the periplasm, recombinant RNase forms the disulfide bridges that are necessary for the enzyme to be fully active. Indeed, the activity of the recombinant RNase is equivalent to that of the commercial enzyme. (3) Although the yields are low (0.1 mg/l of culture) as compared to the systems described above (McGeehan and Benner, 1989; Vasantha and Filipula, 1989), our procedure is highly advantageous since there is no need for solubili-

245

zation of inclusion bodies, refolding of the protein or complicated purification methods. The total procedure takes just 48 h from inoculating the medium to obtaining pure and fully active recombinant RNase. (4) This system is currently in use for the expression of RNase mutants (manuscript in preparation). We have occasionally seen higher levels of expression for one of the mutants although this is not consistent. The presence of RNase I in the periplasm of E. coli (Meador et al., 1990) does not allow the determination of specific activity on the initial periplasmic extract and the reason for variable protein yield is not yet clear. The endogenous E. coli RNase I is separated from recombinant RNase on the Mono-S column. Strategies to increase the yield are underway in order to make crystals of the recombinant enzyme and its variants. (5) The procedure for producing RNase described in this report can be employed for a rapid assessment of the properties of mutant RNases generated by site-directed mutagenesis towards different dinucleotide substrates. These findings will help to elucidate the mechanisms behind the variable substrate specificity of RNase towards dinucleotide substrates.

intervening

sequence

moter like element. Eftink,

M.R.

in the 5’ untranslated Nucleic

and Billonen,

studied

endoribonuclease.

(Eds.),

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a pro-

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V.J. and Kennell,

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We thank Dr. B. Coles for the N-terminal sequencing of the recombinant enzyme, Carol French for the details of the periplasmic release procedure and Elena Sanchez for her help in the early stages of the project. We are also indebted to Dave Marballie for his assistance in the final script. This work was carried out under the support of the procurement executive of the Ministry of Defence.

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utilizing

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for the quantitation the principle

least-

of mi-

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of the bovine pancreatic

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J.: Improved

Ml3

cloning vectors and host strains: nucleotide sequences M13mp18 and pUC19 vectors. Gene 33 (1985) 103-119.

phage of the