Báo cáo Y học: Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology docx

Phages display-ing peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system us

Substrate specificity of human kallikrein 2 (hK2) as determined

by phage display technology

Sylvain M Cloutier1, Jair Ribeiro Chagas2, Jean-Pierre Mach3, Christian M Gygi1, Hans-Jurg Leisinger1 and David Deperthes1

1

Urology Research Unit, Department of Urology, Lausanne, Switzerland;2Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes, Brazil;3Institute of Biochemistry, University of Lausanne, Switzerland

Human glandular kallikrein 2 (hK2) is a trypsin-like serine

protease expressed predominantly in the prostate

epithe-lium Recently, hK2 has proven to be a useful marker that

can be used in combination with prostate specific antigen

for screening and diagnosis of prostate cancer The cleavage

by hK2 of certain substrates in the proteolytic cascade

suggest that the kallikrein may be involved in prostate

cancer development; however, there has been very little

other progress toward its biochemical characterization or

elucidation of its true physiological role In the present

work, we adapt phage substrate technology to study the

substrate specificity of hK2 A phage-displayed random

pentapeptide library with exhaustive diversity was

gener-ated and then screened with purified hK2 Phages

display-ing peptides susceptible to hK2 cleavage were amplified in

eight rounds of selection and genes encoding substrates

were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluores-cent protein) that enables rapid determination of specificity constants This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P¢1 position The scissile bonds identified by phage display substrate selection correspond to those of the nat-ural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development

Keywords: cyan fluorescent protein; human kallikrein; phage display; prostate cancer; substrate

The human prostatic kallikreins hK3, or prostate specific

antigen (PSA), is considered the gold standard for prostate

cancer diagnosis and screening; however, hK2, the second

prostatic kallikrein to be discovered [1], has recently

emerged as a complementary marker for its positive

correlation with prostate cancer grade and progression

PSA is more highly expressed in benign hyperplasia (BHP)

than in cancer thus hK2 is helpful to further distinguish

malignant from benign disease [2–4] The recent discovery

of 12 new members of the kallikrein family [5–7] could

provide additional prostate cancer markers

In the seminal plasma, hK2 is mostly recovered

com-plexed with protein C inhibitor [1] Because hK2 cleaves,

with trypsin-like specificity, certain components of the

semen coagulum (fibronectin and semenogelins), it is

possible that it has a role in the early stages of semen

liquefaction, a biological process which immediately follows

ejaculation [8] In addition, in vitro studies have shown that

hK2 can activate urokinase-type plasminogen activator [9]

and inactivate plasminogen activator inhibitor-1 [10] leading

to the activation of urokinase system Moreover, hK2

degrades insulin-like growth factor binding proteins (IGF-BP) to release IGF, a putative local mitogenic signal for prostate cancer cells [11]

Despite the in vitro identification of its proteolytic activities as well as its potential substrates, our understand-ing of the true physiological role of hK2 remains sketchy Much progress has been made toward the characterization

of hK2s serine protease activity using synthetic substrates derived from reactive serpin loops [12]; however, this type of approach is limited to known targets and cannot advance the discovery of new biological substrates

A system using a monovalent phage library capable of displaying several million different substrates, which enabled simultaneous testing of proteolytic specificity, was developed by Matthews and Wells [13] Several proteases including furin [14], PSA [15], membrane type-1 matrix metalloproteinase [16], and granzyme B [17] have already been characterized using this approach

We adapted this method by constructing a phage-displayed random library that included all possible amino acid combinations of pentapeptides, then screening it with hK2 Of the 44 individual phage clones selected and identified, 90% had Arg at the P1 site and 30% had Ser

in the P¢1 position Kinetic studies and sites of cleavage in substrates have been determined with a new system using cyan fluorescent protein (CFP), a variant of the green fluorescent protein system A search in the SwissProt database with selected substrates identified three new putative hK2 substrates: ADAM-TS8 precursor, cadherin-related tumour suppressor homologue precursor, and collagen alpha (IX) chain precursor

Correspondence to D Deperthes, Urology Research Unit,

Department of Urology, CHUV, CH-1011 Lausanne, Switzerland.

Fax: + 41 213142985, Tel.: + 41 213140120,

E-mail: david.deperthes@urology-research.ch

Abbreviations: PCI, protein C inhibitor; PSA, prostate specific antigen;

CFP, cyan fluorescent protein; IPTG, isopropyl thio-b- D -galactoside.

(Received 23 January 2002, revised 19 April 2002,

accepted 19 April 2002)

M A T E R I A L S A N D M E T H O D S

Materials

Following known methods, hK2 was purified from human

semen [18]; its active site was titrated using

4-methylumbelli-feryl-4-guanidinobenzoate [19] The following materials

were obtained from commercial sources: restriction enzymes

(Roche Biosciences; Amersham Pharmacia), PWO DNA

polymerase and shrimp alkaline phosphatase (Roche

Bio-sciences), T4 DNA ligase (Invitrogen), T4 polynucleotide

kinase (Promega), Ni2+-nitrilotriacetic acid agarose,

anti-His antibody, Ni2+-nitrilotriacetic acid magnetic agarose

beads and 96-well magnet type A (Qiagen) Mycrosynth

GmbH carried out DNA sequencing and oligonucleotides

synthesis

Construction of the substrate phage display library

Substrate phage libraries were generated using a modified

pH0508bphagemid [20] The construction consists of a His6

tag at either end of a Gly-Gly-Gly-Ser-repeat-rich region

that precedes the carboxyl-terminal domain (codons 249–

406) of the M13 gene III The random pentamers were

generated by PCR extension of the template

oligonucleo-tides with appropriate restriction sites positioned on both

side of the degenerate codons: 5¢-TGAGCTAGTCTAGAT

AGGTGGCGGTNNSNNSNNSNNSNNSGGGTCGAC

GTCGGTCATAGCAGTCGCTGCA-3¢ (where N is any

nucleotide and S is either G or C) using 5¢ biotinylated

primers corresponding to the flanking regions: 5¢-TGAGC

TAGTCTAGATAGGTG-3¢ and 5¢-TGCAGCGACTGC

TATGA-3¢ PCR templates are digested and purified as

described previously [21], inserted into XbaI/SalI digested

pH0508bvector, and electroporated into XL1-Blue (F–)

The extent of the library was estimated from the

transfor-mation efficiency determined by plating a small portion of

the transformed cells onto Luria–Bertani plates containing

ampicillin and tetracycline (100 and 15 lgÆmL)1,

respect-ively) The rest of the transformed cells were used to prepare

a phage library by incubating overnight by adding an

M13K07 helper phage at a concentration giving a

multipli-city of infection of 100 plaque forming units (p.f.u.) per mL

Phages were collected from the supernatant and purified by

poly(ethylene glycol) precipitation Of these, 200 clones were

selected arbitrarily for sequencing to verify the

randomiza-tion of the library

Phage-displayed pentapeptide library screening

This new pentapeptide library was subjected to eight

rounds of screening with hK2 One hundred microliters of

Ni2+-nitrilotriacetic acid coupled to sepharose beads

(Ni2+-nitrilotriacetic acid resin) was washed with 10 mL

NaCl/Pi containing 1 mgÆmL)1 BSA Phage particles

(1011) were added to the equilibrated Ni2+-nitrilotriacetic

acid resin and allowed to b ind with gentle agitation for

3 h at 4C The resin was subsequently washed (NaCl/Pi/

BSA 1 mgÆmL)1, 5 mM imidazole, 0.1% Tween 20) to

remove unbound phages and then equilibrated in NaCl/

Pi The substrate phage was exposed to 27 nM (final

concentration) of hK2 for 45 min at 37C A control

selection without protease was also performed The

cleaved phages released into the supernatant were ampli-fied using XL1-Blue Escherichia coli and then used for subsequent rounds of selection After eight rounds of panning, about 15 individual clones were picked from the fifth, sixth and eighth round of selection and plasmid DNA were isolated and sequenced in the region encoding for the substrate

Expression of CFP fluorescent substrate The construction CFP-X5-His contains the following amino acid sequences at the C-terminus of CFP fluorescent proteins: IGGGXXXXXGSTGGGSHHHHHH The ran-dom substrate sequence (in bold) takes place between a His6 tag (underlined) and the CFP protein, separated by the same linker as described previously for substrate phage library The BamHI and XbaI/HindIII DNA recognition sites were introduced by PCR onto 5¢ and 3¢ ends, respectively, of the cDNA encoding the CFP fluorescent protein The PCR product was subcloned into a pQE-16 (Qiagen) vector A DNA duplex encoding the SalI recognition site, the linker, and the His tag was then inserted into the XbaI/HindIII digested vector The resulting CFP-X5-His constructions were used to insert 30 randomly selected substrate genes directly excised from the phage using the XbaI and SalI recognition sites (Fig 1)

In addition, two additional recombinant CFP–X5-His proteins harbouring a peptide known to be either resistant (IKFFS) or sensitive (TFRSA) to hK2 cleavage [12] were constructed and named CFP–Rst and CFP–protein C inhibitor (PCI), respectively To produce recombinant proteins, XL1-Blue cells were transformed with the corres-ponding constructions followed by growth in 50 mL

2· TY (16 g tryptone, 10 g yeast extract, 5 g NaCl per L) with ampicillin (100 lgÆmL) and tetracycline (15 lgÆmL) antibiotics Cells were then induced until D600¼ 0.5 to express recombinant fluorescent substrate by addition of

1 mMof isopropyl thio-b-D-galactoside (IPTG) for 16 h at

37C After an additional 16 h of growth, the cells were harvested by centrifugation and resuspended for 2 h at room temperature in 6 mL denaturation buffer (6MGdN– HCl in NaCl/Piat pH 8.0 containing 10 mM 2-mercapto-ethanol) to recover the soluble and insoluble fractions All recombinant CFPs were purified in denaturing conditions

to prevent substrate cleavage by endogenous bacterial proteases After centrifugation, 100 lL Ni2+-nitrilotriacetic acid resin was added to the bacterial cell supernatant and incubated to bind recombinant proteins The resin was subsequently washed with 5Murea in NaCl/Piat pH 8.0 containing 30 mMimidazole and 10 mM2-mercaptoethanol and proteins were eluted with the same buffer containing

150 mM imidazole The purified recombinant CFPs were diluted 100· in refolding buffer (0.15M Tris/HCl at

pH 8.3, containing 0.1MNaCl and 1 mM 2-mercaptoeth-anol) and the time course of refolding was followed by monitoring increasing fluorescence with a FLX800 fluores-cence 96-well microplate reader, with excitation at 440 nm and emission at 485 nm Once refolding was completed, recombinant CFPs were dialysed against refolding buffer for 14 h at 4C The purity of each refolded proteins was analysed by SDS/PAGE [22] followed by Coomassie Blue staining and Western blot using a horseradish peroxidase-conjugated anti-His Ig (Qiagen)

Direct determination of thekcat/Km

using CFP fluorescent substrates

Refolded CFP-X5-His proteins were fixed to Ni2+

-nitrilo-triacetic acid magnetic beads for 2 h at room temperature

and an aliquot was collected for eluting proteins to

determine the specific activity (fluorescence/amount of

protein) and initial substrate concentration [S0] for each

CFP Concentrations were determined by Bradford assay

(Biorad, USA) All of the kinetic assays were carried out at

37C in 50 mMTris/HCl buffer pH 8.3, containing 0.01%

Tween 20, for 120 min The time course of substrate

hydrolysis was followed by monitoring the fluorescence

released from the beads as the CFPs were cleaved in their

substrate linker Percentage of hydrolysis was calculated as

the ratio of released CFPs to the initial amount of CFPs

bound to the beads which was quantified by elution with

imidazole Specificity constants (kcat/Km) were determined

under pseudo-first order conditions using a substrate

concentration well below the Km[23] Briefly, scissile bonds

in substrates were identified by N-terminal sequencing of

fragments remaining bound to the beads after complete

hydrolysis The final concentration of hK2 was 19 nMfor each enzymatic reaction

R E S U L T S

Construction of the substrate phage library The pH0508bmonovalent phage vector [20] was modified

to generate a new pentamer substrate library with a His tag

at the N-terminus of the random pentapeptides fused to the minor coat protein pIII In this way, the phage can be attached through binding to an immobile phase, in this case the Ni2+-nitrilotriacetic acid resin The constructed library contained 1.8· 108 independent transformants and could thus be considered complete because, in theory, all of the 3.2· 106possible random pentamer sequences were repre-sented The sequencing of phages further confirmed the randomness of the pentamer inserts

Random selection of hK2 substrates Although the filamentous phages are considered to be generally protease resistant, we first verified that hK2 activity had indeed no effect on infectivity Following eight rounds of exposure to hK2, 44 individual phage clones were selected from different rounds; the deduced amino acids corresponding to the substrate sequences are shown in Table 1 No phage was selected more than once, indicating that a large repertoire of susceptible substrates was present

in the pentamer library DNA sequence analysis reveal that

an arginine appears in 40 clones at the P1 site and only one peptide is cleaved at a lysine Among the substrates hydrolysed at an arginine, 11 different amino acids appeared

at the P¢1 subsite However, some amino acids were more frequently recovered at this position with 30% of selected peptides exhibiting serine and 12% methionine, alanine, or valine Interestingly, an evolution of the representation of scissile bonds emerged during the selection (Fig 2); the highest variation was observed for the Arg–Ser scissile bond with continuously increasing recovery of 14, 32, and 42%, respectively, for the fifth, sixth, and eighth rounds of selection A slight increase was also observed in the Arg– Met and Arg–Ala motif, while an important decrease was observed for the Arg–Val motif through the selection, which completely disappeared after eight rounds The positions surrounding the scissile bond at the P3, P2, and P¢2 sub sites predominantly favoured small or uncharged residues as seen

by the 65, 55, and 70% recovery (Fig 3) Of these small or uncharged residues, none in particular was observed more frequently at these positions Hydrophobic residues also appeared in the P3 and P2 subsites in 20% of peptides whereas no aromatic residues were recovered in the P3 and P¢2 positions

CFP fluorescent substrate assay

A simple and direct system has been developed to determine the kinetics of peptide substrate selection from

a phage display library (Fig 1) All CFP recombinant proteins can be produced with good yields in bacteria (1 mg per 50 mL of culture) with 75% being refolded in stable conformations To generate the substrate phage, the CFP–substrate molecule is attached by a His tail to Ni2+

-Fig 1 Schematic outline of the approach used to select substrates for

kallikrein hK2 (1) Phage displaying random peptides fused to a

his-tidine tail (His) are immobilized on an affinity support (Ni 2+

-nitrilo-triacetic acid sepharose beads) (2) After treatment with kallikrein hK2,

phages expressing sensitive substrates are released from the solid phase,

(3) and are then used to infect F-positive bacteria (4) to be amplified for

a next step of selection (5) Phages from the last round of selection are

cloned by plating onto Petri dishes (6) and DNA of individual phages

are amplified in region encoding for the substrate to determine the

sequences cleaved by the enzyme (7) Gene encoding the random

substrate was subcloned into an expression vector, in order to be

produced as a fusion protein between the CFP protein and a histidine

tag (8) The CFP-X5-his protein was fixed to Ni 2+ -nitrilotriacetic acid

magnetic beads and (9) treated by the protease hK2 The released CFP

fluorescent protein was measured with a fluorescence reader (10) which

permitted to determine the percentage of hydrolysis, the specificity

constant and the site of cleavage (11).

nitrilotriacetic acid beads; the substrate can then be released

by hydrolysis only By using two CFP–substrates

harbour-ing a substrate that is either cleavable or resistant to hK2,

we showed that the CFP recombinant protein is cleaved

only in the substrate region and not within the CFP

sequence as no fluorescence was detectable with

CFP-resistant On the other hand, CFP–PCI was efficiently

cleaved with a first-order curve for the product generation

(data not shown) and the specificity constant kcat/Kmwas

 20 000M )1Æs)1

Under the same conditions, hK2 cleaved the other 30

peptides constructed as CFP–substrates with catalytic

efficiencies (kcat/Km) ranging from 1.7· 104

M )1Æs)1 for LRSRA to 9.9· 101

M)1Æs)1 for peptide ERVSP Thus, there is about a 170-fold difference in the efficacy of

cleavage between the different substrates selected by phage

Table 1 Alignment of translated amino acid sequences of random peptide clones selected by substrate phage display with hK2.

Clone

Scissile

Fig 2 Frequency of selection of the different scissile bonds.

display substrate The best substrate peptide, giving

specif-icity constant approaching the PCI–peptide and a

percent-age of hydrolysis superior to 90%, contained a serine

residue in P¢1 subsite whereas the less sensitive peptide

contained a valine, an observation that correlates with the

evolution of the number of different scissile bonds during

the selection The only peptide cleaved at a Lys had a low

specificity constant and gave a percentage of hydrolysis of

only 20% confirming the preference for arginine in the P1

position No cleavage was observed with the two peptides

that did not contain either arginine or lysine suggesting a

residual background among the selected substrates All

peptides having a kcat/Km superior to 5.7· 103

M )1Æs)1 possess two basic amino acids N-terminal to the scissile

bond except for peptide LRSRA where the second basic

residue was found at P¢2 (Tab le 2)

Comparison with natural substrate

When compared to previously reported substrates for hK2,

the peptides selected here had scissile bonds containing the

Arg–Ser motif, which is the same bond cleaved in PCI, a

natural inhibitor of hK2 found in seminal plasma, as well as

semenogelin I, antithrombin III, and kininogen The Arg–

Thr and Arg–Leu motifs are hydrolysed by hK2 in

semenogelins I and II whereas the Arg–Met motif is cleaved

in the plasminogen activator inhibitor-1 and the Arg–Gln

motif is cleaved in IGF-BP-2 Using each of the 44

pentapeptides substrate sequences, FASTA and BLAST

searches were done to look for new potential human protein

substrates of hK2 (Table 3) Among the 11 identical

matches (data not shown), three putative targets were

identified for hK2: ADAM-TS 8 precursor,

cadherin-rela-ted tumour suppressor homologue precursor, and collagen

(IX) chain precursor matching peptides RGRSE, GVFRS

and PGRAP, respectively

D I S C U S S I O N

A wide variety of critical processes depend on specific

cleavage of targets by different enzymes so an ability to

discriminate among many potential substrates is crucial to

maintaining the fidelity of most biological functions

However, unnatural cleavage can occur through

unpredict-able reactions between protease and substrate provoking unexpected biological events such as degradation of extra-cellular matrix, over-availability of growth factors, or degradation of tumor suppressor proteins In the last

5 years, evidence has been mounting that support a role for hK2 in metastasis and cancer progression by virtue of its

in vitroproteolysis of several biological substrates involved

in cancer biology [8–11] However, further investigation is needed to verify this hypothesis Previously biochemical characterizations were incomplete due to the limit of classical iterative methods using already existing or modified substrates [12,24]

The unbiased approach used in this study clearly defined the preferential recognition sites for hK2 substrate hydro-lysis The phage display substrate technique enables millions

of substrates to be screen simultaneously in a single reaction [13,25] Large biological libraries are constructed by displaying random sequences on the extremities of filamen-tous phages, then amplified and screened toward a protease

to survey rapidly its specificity

Most reports using phage display substrate to character-ize proteases have not reported the extent of diversity of the library used in the screening, this being a direct product of the number of different combinations of amino acids displayed by the phage Cloning substrates comprising more than six residues is limited by transformation efficacy, thus the ability to obtain completely adequate diversity with that number of amino acids is questionable [26]

Phage display does generate libraries that are many times more diverse,however, than those using other methods such

as combinatorial chemistry [17] or immobilized positional peptide libraries [27]

In our experiments, randomised pentapeptides were fused

to a truncated form of g3p to produce a library of 1.8· 108 independent recombinant phages where all possible combi-nations of sequences even the rarest polypeptides, are represented The screening of this library with hK2 showed that no phage was in duplicate which is in contrast to selections with other types of phage display libraries (antibody fragments, ligands, or peptide binders) where selections often identified only the best clones with highest reactivity [28,29] Our results are consistent with other studies using phage display that reported a broad diversity but good enrichment in the selection of specific enzyme substrates [13,15,25]

The determination of the specificity constants of the substrates showed a positive tendency during the selection Most of the better substrates were taken the last rounds However, this does not preclude that bad substrates could

be conserved throughout the screening process despite selection pressure Therefore, selected substrates need to be further tested in other configuration than that of fused to a phage The CFP system developed in the present work enabled direct determination of specificity constants and the site of cleavage of the substrate selected by phage display, an improvement over the previously described semiquantitative method [13,25] and chemical synthesis of substrates [15,30] The effectiveness of our system was validated through a recombinant CFP carrying a PCI-derived peptide, a substrate efficiently cleaved by hK2 The kcat/Km of the peptide fused to CFP was significantly lower than that obtained with the same sequence as synthetic fluorogenic form [12]; this difference could be explained by the

Fig 3 P3-P¢2 substrate specificity profile of hK2 from selected peptides

tested as CFP fusion protein Alignment of translated amino-acid

sequences of random peptide clones selected by substrate phage display

with hK2.

modification in the Km caused by the peptide being linked

to a fairly large protein (30 kDa) In addition, hydrophobic

fluorophores used to make intramolecularly quenched

fluorogenic substrates are known to modify the affinity of

peptide for the active site of enzyme, increasing the Km [31]

Our results showing that hK2 cleaves quite selectively

after an arginine residue, concurs with previous reports

[12,24] Nearly one-third of all selected peptides are cleaved

at the Arg–Ser bond despite the large variety of residues

being recovered at the P¢1 position This result shows that

hK2 can accommodate a broad range of amino acids,

except for basic residues, in the P¢1 position The strong

preference for small or noncharged residues is also observed

in P3, P2, and P¢2 subsites but no consensus could be

deduced among the amino acids from the selected sequences Despite this observation, hK2 seems to be dependent on a more extended site of binding than R–S bond for an efficient catalysis as some Arg–Ser peptides possess lower specificity constants Nonetheless, the observation that the best three peptides are cleaved as efficiently as the sequence of PCI– peptide obtained by the classic iterative methods indicates the impressive ability of substrate phage technology to elucidate optimal subsite occupancy for proteases from large banks of randomly selected candidates

Interestingly, the Arg–Ser scissile bond found in numer-ous natural substrates like PCI, semenogelins I and II, fibronectin and kininogen as well as other preferential cleavage sites like Arg–Thr or Arg–Met in seminal coagu-lum proteins and in plasminogen activator inhibitor-1, respectively; is also preferentially selected by hK2 using phage display substrates thus confirming the success of phage display substrate selection

Finally, a SwissProt database search with selected sequences identified three potential human protein sub-strates for hK2 Regions identified in different subsub-strates are extracellular and thus accessible to proteases These poten-tial substrates are not yet well characterized, but are suspected to be involved in cancer progression For example, the desintegrin-like and metalloprotease domain with thrombospondin type I modules 8 (ADAM-TS8)

Table 2 Comparaison of specificity constant (k cat /K m ) values and the percentage hydrolysis of CFP-X5-his based on selected substrates with hK2 (Scissile bonds are designated by fl.)

Table 3 Identification of potential physiological substrate of hK2 using

the SwissProt data base.

HK2 selected

peptides Sequences Potential protein substrate (residues)

8.3 RGRflSE ADAM-TS 8 precursor (646–50)

6.19 GVFRflS Cadherin-related tumour suppressor

homologue precursor (2473–77) 8.17 PGRflAP Collagen alpha (IX) chain precursor

(753–57)

could act as a tumour suppressor through its antiangiogenic

activity [32,33] Cadherin-related tumour suppressor

homo-logue precursor [34] and collagen alpha (IX) chain

precur-sor, a minor cartilage nonfibrillar collagen associated with

type II collagen fibrils [35], are the two other potential

protein substrates for hK2 that could also have a role in

cancer progression

In conclusion, we developed an effective phage display

system that enabled rapid and fruitful investigation of hK2

substrate specificity This powerful technology could

advance the design and the optimization of selective

inhibitors for cancer chemotherapy as well as accelerate

the discovery of new targets Phage display has already

opened new avenues in kallikrein research that may further

reinforce the role of hK2 in the progression of prostate

cancer

A C K N O W L E D G E M E N T S

This work is supported by a grant from the Ligue Suisse contre le

Cancer We thank H Lowman from Genentech Inc for giving

phagemid and fruitful advices.

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