Phage Display Vectors

A large number of different phage display vectors have been constructed. With pretending to be complete, Table 1 lists a selection of phage display vectors. Some of them have not been used for the construction of a library up to now but have been included because they offer possible alternatives. For example, one of the systems allows the success of antibody gene cloning to be monitored by the expression of green fluorescent protein (52).

A variety of different promoters have been used for the expression of antibody genes. Widely used is the lacZ promoter (lacZ) derived from the lactose operon (53). The gIII promoter (gIII) from the bacteriophage M13 (9), the tetracycline promoter (1X teto/p; ref. 54) and the phoA promotor of the E. coli alkaline phosphatase (47) also have been used successfully. It appears that very strong promoters, for example, the synthetic promoter PAI/04/03 (55), are

Phage Display Vectors for Cloning of Antibody Genes in Alphabetical Order

Phage display vector

Promoter

Secretion

Antibody format used by reference

C-domains ir vector

Sites heavy chaii

Sites light chain

Tags

gill

Expression of soluble Ab

Reference

pAALFab

1x lacZ, 2x RBS

2x pelB

Fab

no

EcoR\ - BstP\

Spel - Xho\

truncated

subcloning

77

pAALFv

1x lacZ, 2x RBS

2x pelB

Fv

no

EcoR\ - BstP\

Spe I - Xhol

truncated

subcloning

pAALSC

1x lacZ, 1X RBS

1x pelB

scFv

no

EcoR\ - BstP\

Spe I - Xhol

truncated

subcloning

pAK100

1x lac Z, 1x RBS

1x pelB

scFv

no

SfiI (tet resistance will be removed)

FLAG, myc

truncated

amber, supE strain

78

pAPIII6 scFv

IxphoA, 1x RBS

1 x OmpA

scFv

no

Hindi 11 - Sa/I

FLAG, His

truncated

Sa/I - Kl digest, deletion of gill

79

pCANTAB3his

1x lacZ, 1X RBS

1xg3p

scFv

no

NcoUSfi\ - A/oil

His, myc

full

amber, supE strain

59

pCANTABShis/ pCANTAB6

1x lac Z, 1x RBS

1x cat

scFv

no

NcoUSfi\ - A/oil

His, myc

full

amber, supE strain

pCANTAB 5 E

1x lacZ, 1x RBS

1xg3p

scFv

no

Sfi\ - Noil

E tag

full

amber, supE strain

www.amershamt osciences.com

pCES1

1x lacZ, 2x RBS

1x gill (L) 1 x pelB (H)

Fab

yes

Sfi\ - PstUBstEH (VH)

ApaLA ->4scl (L chain), ApaLI -Xho\ (VU

His, myc

full

amber, supE strain

73

pComb3

2x lac Z, 2x RBS

2x pelB

Fab

no

Xho\ - SpeI

Sacl - Xbal

truncated

Nhe 1 - Spel digest deletion of gill

11

pComb3H

1x lac Z,2x RBS

ompA (LC) pelB (HC)

Fab,scFv

yes

Xho\ - Spe I

Sacl - Xbal

truncated

Nhe\ - Spe 1 digest deletion of gill

80

pComb3X

1x lac Z, 2 x RBS

ompA (LC) pelB (HC)

Fab,scFv

yes

Xho\ - Spe I

Sacl - X6al

His, HA

truncated

amber, supE strain

pCW93/H, pCW99/L1

1x lacZ, 1 x RBS

1x pelB

scFv

no

Nco\ - Nhe\

Sacl - BglH

myc

truncated

amber, supE strain

81

pDAN5

1x lac Z, 1x RBS

undiscribed leader

scFv

no

Xho\ - Nhe I

BssHW - Sa/I

SV5, his

full

amber, supE strain

16

pDH188

2x phoA, 2x RBS

2x stll

Fab

no

n.d.

n.d.

truncated

subcloning

47

pDN322

1x lacZ, 1 x RBS

1x pelB

scFv

no

Ncol - Not\

FLAG, His

full

amber, supE strain

82

pDNEK

1x lac Z, 1 x RBS

1x pelB

scFv

no

Nco \ — Not\

FLAG, His

full

amber, supE strain

83

pEXmide3

1x lacZ, 2x RBS

2x pelB

Fab

yes

SfiUNcoi -Kpn\IApa\

Ea$iNoti -NheUSpel

full

amber, supE strain

84

pEXmide4

1x lacZ, 1 x RBS

1x pelB

scFv

CH1

Ned - Sah

full

amber, supE strain

85

pEXmide5

1x lac Z, 1x RBS

1x pelB

scFv

?

Ned - Sah

full

amber, supE strain

86

pFAB4

2x lacZ, 2x RBS

2x pelB

Fab

no

Sfi'll - Not\

truncated

amber, supE strain

87

pFAB4H

1x lacZ, 2x RBS

2x pelB

Fab

CH1

SfiU - Not\

truncated

subcloning

pFAB5c

2x lac Z, 2x RBS

2x pelB

Fab

no

Sfil

Not\

truncated

amber, supE strain

87

pFAB5c-His

1 x lac Z, 2x RBS

2x pelB

scFv

no

Sfil

Not\

his

truncated

amber, supE strain

89

RBS 1x lac Z, 1x RBS

2x pelB 2x pelB

Fab Fab

CH1 CH1

chain) Sfil - AscI (L chain)

his his

truncated truncated

EagI digest, deletion of gill Eag\ digest, deletion of gIII

91

PGP-F100

1xtet°/p, 1x RBS

1x pelB

scFv

no

Sfi'l (GFPuv will be removed)

myc

truncated

TEV protease site

52

pGZ1

1 x tet°'p, 1x RBS

1x pelB

scFv

no

Sfil

Not\

myc

full

amber, supE strain

54

pHEN1

1 x lac Z, 1 x RBS

1x pelB

scFv, Fab, Fd, LC

no

Sfil

Not\

myc

full

amber, supE strain

14

pHEN1-\^3

1x lac Z, 1 x RBS

1x pelB

scFv

no

A/col - Xho\

\A3 anti-BSA Ab chain

myc

full

amber, supE strain

92

pHEN2

1x lac Z. 1 x RBS

1 x pelB

scFv

no

A/col - Xho\

ApaU - A/oil

his, myc

full

amber, supE strain

http://www.mrc-cpe.cam.ac.uk

pHENIX

1x lac Z, 1 x RBS

1 x pelB

scFv

no

Sfi\/Nco\ -SalUXho\

ApaL 1 - Not\

myc

full

amber, supE strain

93

pHG-1m/A27JKl

1x lac Z, 1x RBS

1 x pelB

scFv

no

ApaU - Sfil

A27JK1 (VL)

his, myc

full

amber, supE strain

94

phh3mu-»/1

2x lac Z, 2x RBS

2x pefB

Fab bidirectional

yes

Xho\ - EcoR\

Sacl - Hindi»

truncated

subcloning

95

plG10

1 x lac Z, 1 x RBS

1x OmpA

scFv

no

EcoRV

- EcoR\

myc

full

amber, supE strain

96

plGT2 (vector)

1 x lac Z, 1 x RBS

1xg3p

scFv

no

Sfil-

A/oil

myc

full

amber, supE strain

50

plGT3 (vector)

1 x lac Z, 1 x RBS

1xg3p

scFv

no

Sfi I

Sfil

myc

full

subcloning

plT2

1 x lac Z, 1 x RBS

1 x pelB

scFv

no

Sfil/A/coI - Xftol

SalL\ - Not\

His, myc

full

amber, supE strain

97

pLG18

1 x phoA, 2x RBS

2x stll

Fab

yes

BssHW - Wcol (CDR2-3)

BstEU - Asp718 (CDR1-3)

truncated

subcloning

98

pM834, pM827

2x lac Z, 2x RBS

2x pelB

Fab

no

Xho\ - Spe I

Sacl - X£>al

full

amber, supE strain

99

pMorph series

1x lac Z ?

1x phoA

scFv

no

Xba\ -

EcoRi

FLAG ?

?

subcloning

100

pScUAGAcp3

1 x lac Z, 1 x RBS

1x pelB

scFv with Ck

Ck

XhoI - Nhe I

Sst\ - Bgftl

truncated

amber, supE strain

101

pSEX

1x PA 1/04/03 1 x RBS

1x pelB

scFv

no

Yol1/34

full

subcloning

12

pSEX20

1x PA 1/04/03 1 x RBS

1 x pelB

scFv

no

YoH/34

full

subcloning

75

pSEX81

1 x lac Z, 1 x RBS

1 x pelB

scFv

no

Nco\ - HincftU

Mlu\ - Not\

Yol1/34

full

subcloning

74

1Cre/lox recombination. 2X recombination. 3Construction of the HuCAL library is described, but the pMorph vectorsystem is unpublished.

N vo rather a disadvantage. To our knowledge, a systematic comparison of the different promotors has not been conducted to date.

The targeting of antibodies to the periplasm requires the use of signal peptides. The pelB leader of the pectate lyase gene of Erwinia carotovora (56) is commonly used. The gill leader (9), the phoA leader of the E. coli alkaline phosphatase, and the ompA leader of E. coli outer membrane protein OmpA have also been used, being common to many protein expression vectors (57,58). Further examples are the heat-stable enterotoxin II (stII) signal sequence (47) and the bacterial chloramphenicol acetyltransferase (cat) leader (59).

Because of the general inability of E. coli to assemble complete IgG, with only one exception documented (60), smaller antibody fragments are used for display. In particular, fragment antigen binding (Fabs) and single-chain Fvs (scFvs) have been shown to be the antibody fragments of choice. In Fabs, the fd fragment and light chain are connected by a disulfide bond. In scFvs, the VH and VL are connected by a 15-25 amino acid linker (61-63). Soluble scFvs tends to form dimers, in particular when the linker is reduced to 3 to 12 amino acid residues. Without or with only a few amino acid linker, diabodies or tetrabodies can be found, with high variability in the behaviour of different V regions (refs. 64-66, Schmiedl and Dübel, unpublished). The dimerization can cause problems with binding because of possible avidity effects of the antibody complex

(67). Furthermore, some scFvs have a reduced affinity of up to one order of magnitude compared with Fabs (62), only in rare cases, scFvs with a higher affinity than the associated Fab have been found

(68). Small antibody fragments like Fv and scFv can easily be produced in E. coli. The yield of functional Fvs expressed in E. coli is higher than the yield of the corresponding Fabs, because of a lower folding rate of the Fabs (69,70). In one example, the stability in long-term storage was much higher for Fabs than for scFvs. After 6 mo, the functionality of scFvs stored at 4°C was reduced by 50%; Fabs, however, showed no significant loss of functionality after 1 yr (71). The overall yield of Fvs expressed in E. coli vary from 0.5 to 10 mg/L culture compared with 2 to 5 mg Fabs/L culture (72).

Therefore, the choice of the antibody fragment, scFv or Fab, depends on the desired application.

For the expression of Fabs in E. coli, two polypeptide chains have to be made. To achieve this, there are two strategies. In the monocistronic systems, for example, pComb3, the antibody genes are under control of two promoters and each has its own leader peptide (11). In plasmids like pCES1 with a bicistronic Fab operon, both chains are under control of a single promoter, leading to mRNA with two ribosomal binding sites (73).

Two variants of pIII-fusions to antibody fragments have been made. Either full-size pIII or a truncated version of pIII have been used. The truncated version is made by deleting the pIII N-terminal domain. This domain mediates the binding to the f-pili of E. coli. Infection is provided by wt pIII because only a small percentage of phage in phagemid-based systems are carrying an antibody. These truncated vectors are therefore not compatible for use with either Hyperphage or Ex-phage, as the full-size pIII is necessary to provide infections (49,50). Some vectors, for example, pSEX81 (74), allow an elution during panning by protease digestion instead of pH shift. This is possible because of a protease cleavage sequence between pIII and scFv and allows a complete recovery of bound phage for infection, even in case of very strong antigen binding.

Most of the described phagemids have an amber stop codon between the antibody gene and gIII. This allows the production of soluble antibodies after transformation of the phagemid to a non suppressor bacterial strain like HB2151 (38). For phagemids like pComb, it is necessary to delete the gIII by digestion followed by religation of the vector before tranformation into E. coli (11). In the case of vectors like pSEX81, the selected antibody genes have to be subcloned into a separate expression vector like the pOPE series (12,75,76).

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