Gene, 101 (1991) 127-131 0

1991 Elsevier

GENE

Science

Publishers

127

B.V. 0378-l 119/91/$03.50

03734

Cloning of coliphage-T4 gene pseT and high-level synthesis of polynucleotide kinase in Esclzericbia cofi (Recombinant

Magalys

DNA;

gene cloning;

expression

plasmid

derivatives;

polymerase

chain reaction;

trp promoter)

Campos”, Mercedes Ortega b, Gabriel Padrlm’, Marion P. Estrada”, Jo& de la Fuente” and Luis Herrera”

u Higher Organisms Genetics Division, b Enzibiot, and ‘Analytical Unit, Centerfor Genetic Engineering and Biotechnology, C.I. G.B., Havana (Cuba) Received by H. Bujard: 9 February Revised: 15 May 1990 Accepted: 29 June 1990

1989

SUMMARY

The gene, pseT, of coliphage T4 which encodes polynucleotide kinase (PNK) was cloned directly into an expression plasmid using the polymerase chain reaction. When placed under the control of the trp promoter, the pseT gene can be maintained stably in Escherichia coli and yields high levels of the enzyme upon induction. The system described facilitates purification and provides very high yields of PNK.

INTRODUCTION

Polynucleotide kinase (PNK) is a product of the coliphage T4 early gene pseT (Sirotkin et al., 1978). It catalyses the transfer of the phosphate of ATP to a 5’-hydroxyl terminus of DNA or RNA (Richardson, 1965). The enzyme also has a 3’-phosphatase activity, which strongly prefers DNA as a substrate (Cameron and Uhlenbeck, 1977; Sirotkin et al., 1978). Due to widespread use of PNK, its gene was cloned in a phage to increase the level of expression in E. colt’ (Midgley and Murray, 1985). The aim of the Correspondence (Cuba)

to: Dr. L. Herrera,

Tel. 20-1401;

Abbreviations:

Ap, ampicillin;

pair(s); d, deletion; chromatography; low melting amide-gel merase

kinase;

fluoride;

SDS,

dodecyl

sodium

encodes

R, resistance/resistant;

Tc, tetracycline;

Tn, transposon;

PAGE,

liquid LMP,

polyacrylPNK, poly-

of E. coli DNA polyPNK;

PMSF,

phenyl-

‘, sensitive/sensitivity;

Sm, streptomycin;

buffer (0.089 M Tris . borate/O.089

bp, base

(medium);

chain reaction;

(large) fragment

T4 gene which sulfate;

albumin;

high-performance

LB, Luria-Bertani

PCR, polymerase

PolIk, Klenow

I; pseT, phage

methylsulfonyl EDTA

serum

HPLC,

oligo, oligodeoxyribonucleotide;

electrophoresis;

nucleotide

P.O. Box 6162, Havana

BSA, bovine

DTT, dithiothreitol; Km, kanamycin;

point;

C.I.G.B.,

Fax 218070.

M boric

TBE, Tris. borateacid/O.002 EDTA);

[ 1, denotes plasmid-carrier

state.

present study was the cloning of the pseT gene and its high-level expression in E. coli, as to facilitate purification and providing high yields of PNK.

EXPERIMENTS

AND RESULTS

(a) Bacterial strains, phages, plasmids, growth conditions and enzymes E. coli strain HBlOl (F-, hsdS20 (rB-, rn,-), supE44, ara-14, galK2, IacYl, proA2, rpsL20 (Sm”), ~~1-5, m&l, A-, recA 13) was used for transformation and analysis of plasmid DNA and E. coli strain JM103 (en&, hsdR, supE, sbcB, thi-1, strA, A(lac-pro), A- [F’traD36, proAB’, Ia0 24151) for sequencing with pBluescript plasmid (Stratagene, U.S.A.). E. coli strain MC1066 hsdR, pyrF74:: Tn5 (KmR), ZeuB6, ara + , trpC9830 A(lacIPOZYA)X74, galU, galK, strAR; HBlOl and W3110 r-m-(~3) F- hsdR_, hsdkf’, [p3(KmR, TcS, Ap’ (tetamR, amp,,R))] for analysis of pseT expression; and E. coli strain LE-392 (F-, hsdR514 (rk- , mk +), supE44, supF58,IacYl or A(IacIZY)6, galK2, galT22, metB1, trpR55, A-) for T4 phage growth. Phage T4 am82 (Panet et al., 1973) was used as a source for the cloning of the pseT gene.

128

The plasmid

Sequencing was carried out according to Chen and Seeburg (1985). DNA was amplified by mixing 10 pg of phage T4

pT5 (Platt et al., 1980) has been described

elsewhere. For plasmid analysis and pseT expression experiments, E. coli cells bearing the plasmid where grown overnight at 37’ C in LB medium supplemented with 50 1.18 Ap/ml. The promoter and Emtage (1980).

was induced

according

DNA in reaction buffer (Saiki et al., 1985) with 100 pmol of each primer (Fig. 1). Samples were subjected to five cycles of PCR, each consisting of 5 min of denaturation at 100’ C, 3 min of cooling to 37 ‘C and 11 min of polymerization after the addition of 1 unit of PolIk. Following ampli-

to Hallewell

All enzymes were obtained from Enzibiot (C.I.G.B., Cuba) and used according to the manufacturer’s recommendations.

fication, the DNA was concentrated by ethanol precipitation and i/lo of the total assay was analyzed by gel electrophoresis

(b) DNA amplification, analysis and cloning Plasmid DNA was prepared according to Birnboim and Doly (1979) and agarose-gel electrophoresis for analysis was performed

as described

by Maniatis

(c) Expression of the pseT gene in Escherichia coli The amplified pseT gene was inserted into pT5 and colonies containing the recombinant plasmid were selected

et al. (1975).

28

q

48

68

8H

82

l.EIEl

128

148

168

1BB

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Primor

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3’

C~~GTCTTCGGG~G~TTTTT~#2TGGCTTGGC CTTC~G~~GCCCTCTfW%WA~TTCWZCGf2~CCG 3’~GCCCTCTWWWI~TT~G 3’

PCR

i‘* 5’ 5’

lmer

1

1 RTG Cla

Pr

--__

STOP

I

E3 Digest

with

Cla

I

*

II m

xx

1 l_iga=r

with

plarmid

pT5