λZLG6: a phage lambda vector for high-efficiency cloning and surface expression of cDNA libraries on filamentous phage

Gene, 173 (1996)179--181 0 1996 Elsevier

Science B.V. All rights reserved.

179

0378-l I19/96/$15.00

09814

GENE

Short Communications

hZLG6: a phage lambda vector for high-efficiency cloning and surface expression of cDNA libraries on filamentous phage (Phage

display; gene VI; selection;

site-specific

recombination)

S. Jespersa,b, Annick De Keysera and Patrick

Laurent

E. Stanssen?*

"Corvas International N. V.. 9000 Gent, Belgium; and bCenter for Transgene Technology and Gene Therapy, Campus Gasthuisherg, 3000 Leuven, Belgium

Received by M.J. Benedik:

10 August

1995; Revised/Accepted:

12 November/24

November

1995; Received at publishers:

11 March

1996

SUMMARY

We describe filamentous

a vector,

phage display

l4. The hZLG6

library

hZLG6,

combining

the high efficiency

of cDNA-encoded is converted

products.

The cDNAs

to a pZLG6-cDNA

infection generates a library of phagemid responding nucleotide sequences within.

particles

of cDNA

are expressed

phagemid

displaying

library

in bacteriophage

display

vectors

based

on gpII1 fusion

for direct

surface

expression

of cDNA

are not libraries

(O’Neil and Hoess, 1995). Display of cDNA products as fusions to the N terminus of this coat protein depends on the presence, in a random primed library, of appropriately 3’-truncated cDNAs that lack the stop codon at the

Recently, phage

h with

as fusions to the 3’ end of Ml3

by in vivo mass excision.

the cDNA-encoded

a functional ideal

cloning

products

Helper

and containing

end of the coding region and are yet capable

INTRODUCTION

Phage

library

gene phage

the cor-

of encoding

product. we have described

display

vector

allowing

an alternative

filamentous

ligand-based

selection

of

cDNAs (Jespers et al., 1995). The system is based on the covalent linkage of cDNA-encoded products to the C terminus

of the Ml3 minor coat protein

VI (gpV1). Three

phagemid vectors (pDONG61, 62 and 63) were derived from pUC119 (Vieira and Messing, 1987) for unidirec-

Correspondence to: Dr. L. Jespers, at his present address: Center for Transgene Technology and Gene Therapy, Campus Gasthuisberg,

tional cloning of cDNA libraries in each reading frame downstream from gene I’Z. Direct fusion of oligo(dT)-

Herestraat

primed

49, 3000 Leuven,

Belgium. Tel. (32-16) 346036; Fax (32-16)

345990; e-mail: [email protected] * Present

address:

The Netherlands. Abbreviations:

Keygene

N.V., P.O. Box 216, 6700 AE Wageningen,

Tel. (31-317) 424141; Fax (31-317) 424939. A, absorbance

(1 cm); Ap, ampicillin;

Cre recombinase

of coliphage

Pl; ELISA,

enzyme-

linked immunosorbent assay; kb, kilobase or 1000 bp; lacZ’, gene encoding the N-terminal end of B-galactosidase; loxP, Cre-specific recombination

site; MCS, multiple

cloning

site; ORF,

open

reading

frame; ori, origin of DNA replication; ori, origin of DNA replication; PCR, polymerase chain reaction; PNPP, p-nitrophenyl phosphate; R, resistance/resistant; TBS, Tris-buffered saline (0.1 M Tris pH 7.4/140 mM NaCl). PII 037X-11

19(96)00217-X

through

practical way to ensure otic host.

their 5’-end represents their

expression

the most

in a prokary-

bp, base pair(s);

CATRIN, Canine Ancylostoma TRypsin INhibitor; cDNA, DNA complementary to RNA; gpIII or gpV1, gene III or VI products of M13; we, gene encoding

cDNAs

EXPERIMENTAL

AND DISCUSSION

(a) Construction of h vectors with excisable phagemid for cDNA display on filamentous phage In order to combine the properties of the pDONG6-vectors with the high efficiency of bacteriophage h for cDNA cloning, we have assembled three h

180 vectors from which gene T/I cDNA expressing phagemids can be excised by an in vivo process. Hogrefe et al. (1993) have described similar h vectors for the cloning and surface display of antibody repertoires fused to the N terminus of gpII1. The h vectors described here are based on hZIPLOX (D’Alessio et al., 1992; Life Technologies, Gaithersburg, MD, USA) which includes a Cre-loxP recombination system (Abremski et al., 1983) enabling automatic phagemid release in Cre-producing DH 12S( ZIP) cells Three separate MunI-Not1 fragments, each containing the Ml3 gene I/I followed by a @I-EcoRI-PmeI-Not1 MCS in a different reading frame, were assembled by PCR technology and inserted within the 1~2’ gene (Fig. 1). The intended h vectors result from the ligation of each MunI-

Not1 fragment with the hZIPLOX/EcoRI left arm and the hZIPLOX/NotI right arm. These vectors allow the directional cloning of cDNA libraries as EcoRI-Not1 (or SJiI-NotI) fragments. The hZLG6 vectors are primarily useful with oligo(dT)-primed cDNA libraries; no stop codons were engineered immediately downstream the Not1 site so as to terminate 3’-truncated ORFs which may occur on random primed cDNAs. (b) Comparison of pZLG6 and pDONG6 phagemid vectors The pZLG6- and pDONG6-type of vectors were compared with respect to their efficiency in directing the surface expression of gene T/I-cDNA fusions. The pDONG6 vectors were previously used to select a cDNA encoding

h ZLG6 1,62 and 63 (40.5 kb) cIts857

nin.5

Sam100

Cre-loxP recombination

’ linkerG

G

MCS

1

I CCTCAGCGGCCGC Not1

Fig. 1. The hZLG6/pZLG6

'

GQVGQNSV* pZLG61

GPSRPESRLNLSGR GGGCCAAGTCGGCCAGAATTCCGTTTAAACCTCAGCGGCCGC NotI PmeI EcoRI

pZLG62

PFKPQRP SAKSARI TCGGCCAAGTCGGCCAGAATTCCGTTTAAACCTCAGCGGCCGC Not1 PmeI EcoRI SfiI

pZLG63

vectors. The three h vectors allow the directional

cloning

of cDNA libraries

in each reading

frame downstream

from the

Ml3 gene VI. The hZLG6 libraries can rapidly be converted in vivo to phagemid libraries (pZLG61, 62 and 63, respectively) by infection of E. coli DHlZS(ZIP) harboring the cre gene on the resident phagemid pZIP (D’Alessio et al., 1992). As the copy number of phagemid rises, the incA locus of pZLG6 inhibits the replication of pZIP by interaction with its Pl ori, leading to a decline in the level of recombinase. The sequence between the EcoRI/MunI ligation point (see section a; underlined), at the junction between lac and Ml3 reads: 5’-GAATTGTTGC TAACATACTG CGTAATAAGG AGTCTTAATC ATG.

sequences,

and the gene VI start codon

(underlined)

181

.

pDONG61 -CATRIP

0

pZLG6 1-CATRIN

0

MI3K07

(c) Conclusions (I) The reported series of h vectors enables efficient cloning of cDNA libraries as fusions to the 3’ end of Ml3 gene I/I. Gene I/I-cDNA expressing phagemid libraries are excised from hZLG6 libraries through Cre-LoxPmediated recombination. Following infection by helper phage, cDNAs can be selected by planning the phage display library against a target such as the natural ligand or an antibody. (2) In an example, the pZLG6 vector was shown to work equally well than the previously reported pDONG6 vector for phage display of cDNA-encoded products.

ACKNOWLEDGEMENT

1o’O Phage/well Fig. 2. Analysis

of pDONG61-CATRIN

phage by ELISA. Streptavidin-coated bated

Tween-10,

Phage

were incubated

was used as negative

phosphatase

serially

trypsin diluted

(biotin-SS-NHS

in TBS containing

in the wells for 2 h. Helper

control.

phage were detected

rum, alkaline PNPP

samples,

After washing by incubation

conjugated

fusion

96 well plates (Pierce) were incu-

with 100 ul of 10 nM biotinylated

gent; Pierce).

bound

and pZLG61-CATRIN

We are grateful to Dr. George Vlasuk for critically reading this manuscript.

rea-

phage M13K07

with TBSjO. 1% Tween-20, with rabbit

anti-rabbit

anti-Ml3

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