Patent Application: US-45047203-A

Abstract:
a novel family of protein libraries comprising ctlds in which internal polypeptide loop - regions lining the ligand binding sites in ctlds have been replaced with ensembles of completely or partially randomised polypeptide segments . tetranectin ctlds were chosen as framework for the preferred embodiment of the invention ; and versatile phagemid vectors useful in the generation and manipulation of human and murine tetranectin ctld libraries are disclosed as part of this invention . tetranectin ctlds in monomeric as well as in trimeric form are efficiently displayed as gene iii fusions in fully functional form by the recombinant fd phage display vector . ctld derivatives with affinity for new ligands may readily be isolated from libraries of vectors displaying ctlds , in which loop - regions have been randomised , using one or more rounds of enrichment by screening or selection followed by amplification of the enriched subpopulation in each round . the efficiency with which protein products containing ctlds with new binding properties can be produced , e . g . by bacterial expression and in vitro refolding , in mono -, tri -, or multimeric formats provides important advantages in terms of simplicity , cost and efficiency of generation , production and diagnostic or therapeutic applications in comparison to recombinant antibody derivatives .

Description:
the terms “ c - type lectin - like protein ” and “ c - type lectin ” are used to refer to any protein present in , or encoded in the genomes of , any eukaryotic species , which protein contains one or more ctlds or one or more domains belonging to a subgroup of ctlds , the crds , which bind carbohydrate ligands . the definition specifically includes membrane attached c - type lectin - like proteins and c - type lectins , “ soluble ” c - type lectin - like proteins and c - type lectins lacking a functional transmembrane domain and variant c - type lectin - like proteins and c - type lectins in which one or more amino acid residues have been altered in vivo by glycosylation or any other post - synthetic modification , as well as any product that is obtained by chemical modification of c - type lectin - like proteins and c - type lectins . in the claims and throughout the specification certain alterations may be defined with reference to amino acid residue numbers of a ctld domain or a ctld - containing protein . the amino acid numbering starts at the first n - terminal amino acid of the ctld or the native or artificial ctld - containing protein product , as the case may be , which shall in each case be indicated by unambiguous external literature reference or internal reference to a figure contained herein within the textual context . the terms “ amino acid ”, “ amino acids ” and “ amino acid residues ” refer to all naturally occurring l - α - amino acids . this definition is meant to include norleucine , or nithine , and homocysteine . the amino acids are identified by either the single - letter or three - letter designations : asp d aspartic acid ile i isoleucine thr t threonine leu l leucine ser s serine tyr y tyrosine glu e glutamic acid phe f phenylalanine pro p proline his h histidine gly g glycine lys k lysine ala a alanine arg r arginine cys c cysteine trp w tryptophan val v valine gln q glutamine met m methionine asn n asparagine nle j norleucine orn o ornithine hcy u homocysteine xxx x any l - α - amino acid . the naturally occurring l - α - amino acids may be classified according to the chemical composition and properties of their side chains . they are broadly classified into two groups , charged and uncharged . each of these groups is divided into subgroups to classify the amino acids more accurately : hydrophilic residues : ser , thr , asn , gln aliphatic residues : gly , ala , val , leu , ile , nle non - polar residues : cys , met , pro , hcy aromatic residues : phe , tyr , trp the terms “ amino acid alteration ” and “ alteration ” refer to amino acid substitutions , deletions or insertions or any combinations thereof in a ctld amino acid sequence . in the ctld variants of the present invention such alteration is at a site or sites of a ctld amino acid sequence . substitutional variants herein are those that have at least one amino acid residue in a native ctld sequence removed and a different amino acid inserted in its place at the same position . the substitutions may be single , where only one amino acid in the molecule has been substituted , or they may be multiple , where two or more amino acids have been substituted in the same molecule . the designation of the substitution variants herein consists of a letter followed by a number followed by a letter . the first ( leftmost ) letter designates the amino acid in the native ( unaltered ) ctld or ctld - containing protein . the number refers to the amino acid position where the amino acid substitution is being made , and the second ( righthand ) letter designates the amino acid that is used to replace the native amino acid . as mentioned above , the numbering starts with “ 1 ” designating the n - terminal amino acid sequence of the ctld or the ctld - containing protein , as the case may be . multiple alterations are separated by a comma (,) in the notation for ease of reading them . the terms “ nucleic acid molecule encoding ”, “ dna sequence encoding ”, and “ dna encoding ” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid . the order of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain . the dna sequence thus encodes the amino acid sequence . the terms “ mutationally randomised sequence ”, “ randomised polypeptide segment ”, “ randomised amino acid sequence ”., “ randomised oligonucleotide ” and “ mutationally randomised sequence ”, as well as any similar terms used in any context to refer to randomised sequences , polypeptides or nucleic acids , refer to ensembles of polypeptide or nucleic acid sequences or segments , in which the amino acid residue or nucleotide at one or more sequence positions may differ between different members of the ensemble of polypeptides or nucleic acids , such that the amino acid residue or nucleotide occurring at each such sequence position may belong to a set of amino acid residues or nucleotides that may include all possible amino acid residues or nucleotides or any restricted subset thereof . said terms are often used to refer to ensembles in which the number of amino acid residues or nucleotides is the same for each member of the ensemble , but may also be used to refer to such ensembles in which the number of amino acid residues or nucleotides in each member of the ensemble may be any integer number within an appropriate range of integer numbers . several systems displaying phenotype , in terms of putative ligand binding modules or modules with putative enzymatic activity , have been described . these include : phage display ( e . g . the filamentous phage fd [ dunn ( 1996 ), griffiths amd duncan ( 1998 ), marks et al . ( 1992 )], phage lambda [ mikawa et al . ( 1996 )]), display on eukarotic virus ( e . g . baculovirus [ ernst et al . ( 2000 )]), cell display ( e . g . display on bacterial cells [ benhar et al . ( 2000 )], yeast cells [ boder and wittrup ( 1997 )], and mammalian cells [ whitehorn et al . ( 1995 )], ribosome linked display [ schaffitzel et al . ( 1999 )], and plasmid linked display [ gates et al . ( 1996 )]. the most commonly used method for phenotype display and linking this to genotype is by phage display . this is accomplished by insertion of the reading frame encoding the scaffold protein or protein of interest into an intra - domain segment of a surface exposed phage protein . the filamentous phage fd ( e . g . m13 ) has proven most useful for this purpose . polypeptides , protein domains , or proteins are the most frequently inserted either between the “ export ” signal and domain 1 of the fd gene iii protein or into a so - called hinge region between domain 2 and domain 3 of the fd - phage gene iii protein . human antibodies are the most frequently used proteins for the isolation of new binding units , but other proteins and domains have also been used ( e . g . human growth hormone [ bass et al . ( 1990 )], alkaline phosphatase [ mccafferty et al . ( 1991 )], β - lactamase inhibitory protein [ huang et al . ( 2000 )], and cytotoxic t lymphocyte - associated antigen 4 [ hufton et al . ( 2000 )]. the antibodies are often expressed and presented as scfv or fab fusion proteins . three strategies have been employed . either a specific antibody is used as a scaffold for generating a library of mutationally randomised sequences within the antigen binding clefts [ e . g . fuji et al . ( 1998 )] or libraries representing large ensembles of human antibody encoding genes from non - immunised hosts [ e . g . nissim et al . ( 1994 )] or from immunised hosts [ e . g . cyr and hudspeth ( 2000 )] are cloned into the fd phage vector . the general procedure for accomplishing the generation of a display system for the generation of ctld libraries comprise essentially ( 1 ) identification of the location of the loop - region , by referring to the 3d structure of the ctld of choice , if such information is available , or , if not , identification of the sequence locations of the β2 -, β3 - and β4 strands by sequence alignment with the sequences shown in fig1 as aided by the further corroboration by identification of sequence elements corresponding to the β2 and β3 consensus sequence elements and β4 - strand characteristics , also disclosed above ; ( 2 ) subcloning of a nucleic acid fragment encoding the ctld of choice in a protein display vector system with or without prior insertion of endonuclease restriction sites close to the sequences encoding β2 , β3 and β4 ; and ( 3 ) substituting the nucleic acid fragment encoding some or all of the loop - region of the ctld of choice with randomly selected members of an ensemble consisting of a multitude of nucleic acid fragments which after insertion into the nucleic acid context encoding the receiving framework will substitute the nucleic acid fragment encoding the original loop - region polypeptide fragments with randomly selected nucleic acid fragments . each of the cloned nucleic acid fragments , encoding a new polypeptide replacing an original loop - segment or the entire loop - region , will be decoded in the reading frame determined within its new sequence context . nucleic acid fragments may be inserted in specific locations into receiving nucleic acids by any common method of molecular cloning of nucleic acids , such as by appropriately designed pcr manipulations in which chemically synthesized nucleic acids are copy - edited into the receiving nucleic acid , in which case no endonuclease restriction sites are required for insertion . alternatively , the insertion / excision of nucleic acid fragments may be facilitated by engineering appropriate combinations of endonuclease restriction sites into the target nucleic acid into which suitably designed oligonucleotide fragments may be inserted using standard methods of molecular cloning of nucleic acids . it will be apparent that interesting ctld variants isolated from ctld libraries in which restriction endonuclease sites have been inserted for convenience may contain mutated or additional amino acid residues that neither correspond to residues present in the original ctld nor are important for maintaining the interesting new affinity of the ctld variant . if desirable , e . g . in case the product needs to be rendered as non - immunogenic as possible , such residues may be altered or removed by back - mutation or deletion in the specific clone , as appropriate . the ensemble consisting of a multitude of nucleic acid fragments may be obtained by ordinary methods for chemical synthesis of nucleic acids by directing the step - wise synthesis to add pre - defined combinations of pure nucleotide monomers or a mixture of any combination of nucleotide monomers at each step in the chemical synthesis of the nucleic acid fragment . in this way it is possible to generate any level of sequence degeneracy , from one unique nucleic acid sequence to the most complex mixture , which will represent a complete or incomplete representation of maximum number unique sequences of 4 n , where n is the number of nucleotides in the sequence . complex ensembles consisting of multitudes of nucleic acid fragments may , alternatively , be prepared by generating mixtures of nucleic acid fragments by chemical , physical or enzymatic fragmentation of high - molecular mass nucleic acid compositions like , e . g ., genomic nucleic acids extracted from any organism . to render such mixtures of nucleic acid fragments useful in the generation of molecular ensembles , as described here , the crude mixtures of fragments , obtained in the initial cleavage step , would typically be size - fractionated to obtain fragments of an approximate molecular mass range which would then typically be adjoined to a suitable pair of linker nucleic acids , designed to facilitate insertion of the linker - embedded mixtures of size - restricted oligonucleotide fragments into the receiving nucleic acid vector . to facilitate the construction of combinatorial ctld libraries in tetranectin , the model ctld of the preferred embodiment of the invention , suitable restriction sites located in the vicinity of the nucleic acid sequences encoding β2 , β3 and β4 in both human and murine tetranectin were designed with minimal perturbation of the polypeptide sequence encoded by the altered sequences . it was found possible to establish a design strategy , as detailed below , by which identical endonuclease restriction sites could be introduced at corresponding locations in the two sequences , allowing interesting loop - region variants to be readily excised from a recombinant murine ctld and inserted correctly into the ctld framework of human tetranectin or vice versa . analysis of the nucleotide sequence encoding the mature form of human tetranectin reveals ( fig2 ) that a recognition site for the restriction endonuclease bgl ii is found at position 326 to 331 ( agatct ), involving the encoded residues glu109 , ile110 , and trp111 of β2 , and that a recognition site for the restriction endonuclease kas i is found at position 382 to 387 ( ggcgcc ), involving the encoded amino acid residues gly128 and ala129 ( located c - terminally in loop 2 ). mutation , by site directed mutagenesis , of g513 to a and of c514 to t in the nucleotide sequence encoding human tetranectin would introduce a mun i restriction endonuclease recognition site therein , located at position 511 to 516 , and mutation of g513 to a in the nucleotide sequence encoding murine tetranectin would introduce a mun i restriction endonuclease site therein at a position corresponding to the mun i site in human tetranectin , without affecting the amino acid sequence of either of the encoded protomers . mutation , by site directed mutagenesis , of c327 to g and of g386 to c in the nucleotide sequence encoding murine tetranectin would introduce a bgl ii and a kas i restriction endonuclease recognition site , respectively , therein . additionally , a325 in the nucleotide sequence encoding murine tetranectin is mutagenized to a g . these three mutations would affect the encoded amino acid sequence by substitution of asn109 to glu and gly129 to ala , respectively . now , the restriction endonuclease kas i is known to exhibit marked site preference and cleaves only slowly the tetranectin coding region . therefore , a recognition site for another restriction endonuclease substituting the kas i site is preferred ( e . g . the recognition site for the restriction endonuclease kpn i , recognition sequence ggtacc ). the nucleotide and amino acid sequences of the resulting tetranectin derivatives , human tetranectin lectin ( htlec ) and murine tetranectin lectin ( mtlec ) are shown in fig3 . the nucleotide sequences encoding the htlec and mtlec protomers may readily be subcloned into devices enabling protein display of the linked nucleotide sequence ( e . g . phagemid vectors ) and into plasmids designed for heterologous expression of protein [ e . g . pt7h6 , christensen et al . ( 1991 )]. other derivatives encoding only the mutated ctlds of either htlec or mtlec ( htctld and mtctld , respectively ) have also been constructed and subcloned into phagemid vectors and expression plasmids , and the nucleotide and amino acid sequences of these ctld derivatives are shown in fig4 . the presence of a common set of recognition sites for the restriction endonucleases bgl ii , kas i or kpn i , and mun i in the ensemble of tetranectin and ctld derivatives allows for the generation of protein libraries with randomised amino acid sequence in one or more of the loops and at single residue positions in β4 comprising the lectin ligand binding region by ligation of randomised oligonucleotides into properly restricted phagemid vectors encoding htlec , mtlec , htctld , or mtctld derivatives . after rounds of selection on specific targets ( e . g . eukaryotic cells , virus , bacteria , specific proteins , polysaccharides , other polymers , organic compounds etc .) dna may be isolated from the specific phages , and the nucleotide sequence of the segments encoding the ligand - binding region determined , excised from the phagemid dna and transferred to the appropriate derivative expression vector for heterologous production of the desired product . heterologous production in a prokaryote may be preferred because an efficient protocol for the isolation and refolding of tetranectin and derivatives has been reported ( international patent application publication wo 94 / 18227 a2 ). a particular advantage gained by implementing the technology of the invention , using tetranectin as the scaffold structure , is that the structures of the murine and human tetranectin scaffolds are almost identical , allowing loop regions to be swapped freely between murine and human tetranectin derivatives with retention of functionality . swapping of loop regions between the murine and the human framework is readily accomplished within the described system of tetranectin derivative vectors , and it is anticipated , that the system can be extended to include other species ( e . g . rat , old and new world monkeys , dog , cattle , sheep , goat etc .) of relevance in medicine or veterinary medicine in view of the high level of homology between man and mouse sequences , even at the genetic level . extension of this strategy to include more species may be rendered possible as and when tetranectin is eventually cloned and / or sequenced from such species . because the c - type lectin ligand - binding region represents a different topological unit compared to the antigen binding clefts of the antibodies , we envisage that the selected binding specificities will be of a different nature compared to the antibodies . further , we envisage that the tetranectin derivatives may have advantages compared to antibodies with respect to specificity in binding sugar moieties or polysaccharides . the tetranectin derivatives may also be advantageous in selecting binding specificities against certain natural or synthetic organic compounds . several ctlds are known to bind calcium ions , and binding of other ligands is often either dependent on calcium ( e . g . the collectin family of c - type lectins , where the calcium ion bound in site 2 is directly involved in binding the sugar ligand [ weis and drickamer ( 1996 )]) or sensitive to calcium ( e . g . tetranectin , where binding of calcium involves more of the side chains known otherwise to be involved in plasminogen kringle 4 binding [ graversen et al . ( 1998 )]). the calcium binding sites characteristic of the c - type lectin - like protein family are comprised by residues located in loop 1 , loop 4 and β - strand 4 and are dependent on the presence of a proline residue ( often interspacing loop 3 and loop 4 in the structure ), which upon binding is found invariantly in the cis conformation . moreover , binding of calcium is known to enforce structural changes in the ctld loop - region [ ng et al . ( 1998a , b )]. we therefore envisage , that binding to a specific target ligand by members of combinational libraries with preserved ctld metal binding sites may be modulated by addition or removal of divalent metal ions ( e . g . calcium ions ) either because the metal ion may be directly involved in binding , because it is a competitive ligand , or because binding of the metal ion enforces structural rearrangements within the putative binding site . the trimeric nature of several members of the c - type lectin and c - type lectin - like protein family , including tetranectin , and the accompanying avidity in binding may also be exploited in the creation of binding units with very high binding affinity . as can be appreciated from the disclosure above , the present invention has a broad general scope and a wide area of application . accordingly , the following examples , describing various embodiments thereof , are offered by way of illustration only , not by way of limitation . construction of tetranectin derived e . coli expression plasmids and phagemids the expression plasmid pt7h 6 fx - htlec , encoding the fx - htlec ( seq id no : 01 ) part of full length h 6 fx - htlec fusion protein , was constructed by a series of four consecutive site - directed mutagenesis experiments starting from the expression plasmid pt7h6 - rtn 123 [ holtet et al . ( 1997 )] using the quickchange ™ site - directed mutagenesis kit ( stratagene , la jolla , calif .) and performed as described by the manufacturer . mismatching primer pairs introducing the desired mutations were supplied by dna technology ( aarhus , denmark ). an outline of the resulting pt7h 6 fx - htlec expression plasmid is shown in fig5 and the nucleotide sequence of the fx - htlec encoding insert is given as seq id no : 01 . the amino acid sequence of the fx - htlec part of the h 6 fx - htlec fusion protein is shown in fig6 and given as seq id no : 02 . the expression plasmid pt7h 6 fx - htctld , encoding the fx - htctld ( seq id no : 03 ) part of the h 6 fx - htctld fusion protein , was constructed by amplification and subcloning into the plasmid pt7h6 ( i . e . amplification in a polymerase chain reaction using the expression plasmid pt7h6 - htlec as template , and otherwise the primers , conditions , and subcloning procedure described for the construction of the expression plasmid pt7h6tn3 [ holtet et al . ( 1997 )]. an outline of the resulting pt7h 6 fx - htctld expression plasmid is shown in fig7 and the nucleotide sequence of the fx - htctld encoding insert is given as seq id no : 03 . the amino acid sequence of the fx - htctld part of the h 6 fx - htctld fusion protein is shown in fig8 and given as seq id no : 04 . the phagemids , pphtn and pphtn3 , were constructed by ligation of the sfi i and not i restricted dna fragments amplified from the expression plasmids pt7h6 - rtn 123 ( with the oligonucleotide primers 5 - cggctgagcggccca - gccggccatggccgagccaccaacccagaagc - 3 ′ [ seq id no : 05 ] and 5 ′- cctgcggccgccacgatcccgaactgg - 3 ′ [ seq id no : 06 ]) and pt7h 6 fx - htctld ( with the oligonucleotide primers 5 ′- cggctgagcggcccagccggccatggccgccctgcagacggtc - 3 ′ [ seq id no : 07 ] and 5 ′- cctgcggccgccacgatcccgaactgg - 31 [ seq id no : 06 ]), respectively , into a sfi i and not i precut vector , pcantab 5e supplied by amersham pharmacia biotech ( code no . 27 - 9401 - 01 ) using standard procedures . outlines of the resulting pphtn and pphtn3 phagemids are shown in fig9 and fig1 , respectively , and the nucleotide sequences of the phtn and phtn3 inserts are given as seq id no : 08 and seq id no : 10 , respectively . the amino acid sequences encoded by the phtn and phtn3 inserts are shown in fig1 ( seq id no : 09 ) and fig1 ( seq id no : 11 ), respectively . the phagemids , pphtlec and pphtctld , were constructed by ligation of the sfi i and not i restricted dna fragments amplified from the expression plasmids pt7h 6 fx - htlec ( with the oligonucleotide primers 5 - cggctgagcggcccagcc - ggccatggccgagccaccaacccagaagc - 3 ′ [ seq id no : 05 ] and 5 ′- cctgcggccgccacgatcccgaactgg - 3 ′ [ seq id no : 06 ]) and pt7h 6 fx - htctld ( with the oligonucleotide primers 5 ′- cggctgagcggcccagccggccatggccgccctgcagacggtc - 3 ′ [ seq id no : 07 ] and 5 ′- cctgcggccgccacgatcccgaactgg - 3 ′ [ seq id no : 06 ]), respectively , into a sfi i and not i precut vector , pcantab 5e supplied by amersham pharmacia biotech ( code no . 27 - 9401 - 01 ) using standard procedures . outlines of the resulting pphtlec and pphtctld phagemids are shown in fig1 and fig1 , respectively , and the nucleotide sequences of the phtlec and phtctld inserts are given as seq id no : 12 and seq id no : 14 , repectively . the amino acid sequences encoded by the phtlec and phtctld inserts are shown in fig1 ( seq id no : 13 ) and fig1 ( seq id no : 15 ), respectively . a plasmid clone , puc - mtlec , containing the nucleotide sequence corresponding to the murine tetranectin derivative mtlec ( fig3 and seq id no : 16 ) was constructed by four succesive subclonings of dna subfragments in the following way : first , two oligonucleotides 5 ′- cggaattcgagtcacccactcccaaggccaagaaggctgcaaatgccaagaaa - gatttggtgagctcaaagatgttc - 3 ′ ( seq id no : 17 ) and 5 ′- gcg - gatccaggcctgcttctccttcagcagggccacctcctgggccaggacatccatcctgttcttgagctcctcgaacatctttgagctcacc - 3 ′ ( seq id no : 18 ) were annealed and after a filling in reaction cut with the restriction endonucleases eco ri ( gaattc ) and bam hi ( ggatcc ) and ligated into eco ri and bam hi precut puc18 plasmid dna . second , a pair of oligonucleotides 5 ′- gcaggccttacagactgtgtgcctgaagggcaccaaggtgaacttgaagtgcctcctggccttcacccaaccgaagaccttccatgaggcgagcgag - 3 ′ ( seq id no : 19 ) and 5 ′- ccgcatgcttcgaacagcgcctcgttctctagctctgactgcggggtgcccagcgtgcccccttgcgagatgcagtcctcgctcgcctcatgg - 3 ′ ( seq id no : 20 ) was annealed and after a filling in reaction cut with the restriction endonucleases stu i ( aggcct ) and sph i ( gcatgc ) and ligated into the stu i and . sph i precut plasmid resulting from the first ligation . third , an oligonucleotide pair 5 ′- ggttcgaatacgcgcgccacagcgtgggcaacgatgcggagatctaaatgctcccaattgc - 3 ′ ( seq id no : 21 ) and 5 ′- ccaagcttcacaatggcaaactggcagatgtagggcaattgggagcatttagatc - 3 ′ ( seq id no : 22 ) was annealed and after a filling in reaction cut with the restriction endonucleases bstb i ( ttcgaa ) and hind iii ( aagctt ) and ligated into the bstb i and hind iii precut plasmid resulting from the second ligation . fourth , an oligonucleotide pair 5 ′- cggagatctggctgggcctcaacgacatggccgcggaaggcgcctgggtggacatgaccggtaccctcctggcctacaagaactgg - 3 ′ ( seq id no : 23 ) and 5 ′- gggcaattgatcgcggcatcgcttgtcgaacctcttgccgttggctgcgccagacagggcggcgcagttctcggctttgccgccgtcgggttgcgtcgtgatctccgtctcccagttcttgtaggccagg - 3 ′ ( seq id no : 24 ) was annealed and after a filling in reaction cut with the restriction endonucleases bgl ii ( agatct ) and mun i ( caattg ) and ligated into the bgl ii and mun i precut plasmid resulting from the third ligation . an outline of the puc - mtlec plasmid is shown in fig1 , and the resulting nucleotide sequence of the eco ri to hind iii insert is given as seq id no : 16 . the expression plasmids pt7h 6 fx - mtlec and pt7h 6 fx - mtctld may be constructed by ligation of the bam hi and hind iii restricted dna fragments , amplified from the puc - mtlec plasmid with the oligonucleotide primer pair 5 - ctgggatccatccagggtcgcgagtcacccactcccaagg - 3 ′ ( seq id no : 25 ) and 5 ′- ccgaagcttacacaatggcaaactggc - 3 ′ ( seq id no : 26 ), and with the oligonucleotide primer pair 5 ′- ctgggatccatccagggtcgcgccttacagactgtggtc - 3 ′ ( seq id no : 27 ), and 5 ′- ccgaagcttacacaatggcaaactggc - 3 ′ ( seq id no : 26 ), respectively , into bam hi and hind iii precut pt7h6 vector using standard procedures . an outline of the expression plasmids pt7h 6 fx - mtlec and pt7h 6 fx - mtctld is shown in fig1 and fig2 , respectively , and the nucleotide sequences of the fx - mtlec and fx - mtctld inserts are given as seq id no : 28 and seq id no : 30 , respectively . the amino acid sequences of the fx - mtlec and fx - mtctld parts of the fusion proteins h 6 fx - mtlec and h 6 fx - mtctld fusion proteins are shown in fig1 ( seq id no : 29 ) and fig2 ( seq id no : 31 ), respectively . the phagemids ppmtlec and ppmtctld may be constructed by ligation of the sfi i and not i restricted dna fragments ( amplified from the puc - mtlec plasmid with the oligonucleotide primer pair 5 - cggctgagcggcccagccggccatggccgagtcacccactcccaagg - 3 ′ [ seq id no : 32 ], and 5 ′- cctgcggccgccacgatcccgaactgg - 3 ′ [ seq id no : 33 ] and with the oligonucleotide primers 5 , - cggctgagcggcccagccggccatggccgccttacagactgtggtc - 3 ′ [ seq id no : 34 ] and 5 ′- cctgcggccgccacgatcccgaactgg - 3 , [ seq id no : 33 ], respectively ) into a sfi i and not i precut vector pcantab 5e supplied by amersham pharmacia biotech ( code no . 27 - 9401 - 01 ) using standard procedures . outlines of the ppmtlec and ppmtctld plasmids are shown in fig2 and fig2 , respectively , and the resulting nucleotide sequences of the pmtlec and pmtctld inserts are given as seq id no : 35 and seq id no : 37 , repectively . the amino acid sequences encoded by the pmtlec and pmtctld inserts are shown in fig2 ( seq id no : 36 ) and fig2 ( seq id no : 38 ), respectively . in order to verify that the phtlec and phtn3 gene iii fusion proteins can indeed be displayed by the recombinant phage particles , the phagemids pphtlec and pphtn3 ( described in example 1 ) were transformed into e . coli tg1 cells and recombinant phages produced upon infection with the helper phage m13ko7 . recombinant phages were isolated by precipitation with poly ( ethylene glycol ) ( peg 8000 ) and samples of phtlec and phtn3 phage preparations as well as a sample of helper phage were subjected to an elisa - type sandwich assay , in which wells of a maxisorb ( nunc ) multiwell plate were first incubated with anti - human tetranectin or bovine serum albumin ( bsa ) and blocked in skimmed milk or skimmed milk / edta . briefly , cultures of pphtlec and pphtn3 phagemid transformed tg1 cells were grown at 37 ° c . in 2 × ty - medium supplemented with 2 % glucose and 100 mg / l ampicillin until a 600 reached 0 . 5 . by then the helper phage , m13k07 , was added to a concentration of 5 × 10 9 pfu / ml . the cultures were incubated at 37 ° c . for another 30 min before cells were harvested by centrifugation and resuspended in the same culture volume of 2 × ty medium supplemented with 50 mg / l kanamycin and 100 mg / l ampicillin and transferred to a fresh set of flasks and grown for 16 hours at 25 ° c . cells were removed by centrifugation and the phages precipitated from 20 ml culture supernatant by the addition of 6 ml of ice cold 20 % peg 8000 , 2 . 5 m nacl . after mixing the solution was left on ice for one hour and centrifuged at 4 ° c . to isolate the precipitated phages . each phage pellet was resuspended in 1 ml of 10 mm tris - hcl ph 8 , 1 mm edta ( te ) and incubated for 30 min before centrifugation . the phage containing supernatant was transferred to a fresh tube . along with the preparation of phage samples , the wells of a maxisorb plate was coated overnight with ( 70 μl ) rabbit anti - human tetranectin ( a polyclonal antibody from dako a / s , code no . a0371 ) in a 1 : 2000 dilution or with ( 70 μl ) bsa ( 10 mg / ml ). upon coating , the wells were washed three times with pbs ( 2 . 68 mm kcl , 1 . 47 mm kh 2 po 4 , 137 mm nacl , 8 . 10 mm na 2 hpo 4 , ph 7 . 4 ) and blocked for one hour at 37 ° c . with 280 μl of either 3 % skimmed milk in pbs , or 3 % skimmed milk , 5 mm edta in pbs . anti - tetranectin coated and bsa coated wells were then incubated with human phtlec -, phtn3 -, or helper phage samples for 1 hour and then washed 3 times in pbs buffer supplemented with the appropriate blocking agent . phages in the wells were detected after incubation with hrp - conjugated anti - phage conjugate ( amersham pharmacia , code no . 27 - 9421 - 01 ) followed by further washing . hrp activities were then measured in a 96 - well elisa reader using a standard hrp chromogenic substrate assay . phtlec and phtn3 phages produced strong responses ( 14 times background ) in the assay , irrespective of the presence or absence of edta in the blocking agent , whereas helper phage produced no response above background readings in either blocking agent only low binding to bsa was observed ( fig2 ). it can therefore be concluded that the human phtlec and phtn3 phages both display epitopes that are specifically recognized by the anti - human tetranectin antibody . demonstration of authentic ligand binding properties of phtlec and phtn3 displayed on phase the apo - form of the ctld domain of human tetranectin binds in a lysine - sensitive manner specifically to the kringle 4 domain of human plasminogen [ graversen et al . ( 1998 )]. binding of tetranectin to plasminogen can be inhibited by calcium which binds to two sites in the ligand - binding site in the ctld domain ( kd approx . 0 . 2 millimolar ) or by lysine - analogues like amcha ( 6 - amino - cyclohexanoic acid ), which bind specifically in the two stronger lysine - binding sites in plasminogen of which one is located in kringle 1 and one is located in kringle 4 ( kd approx . 15 micromolar ). to demonstrate specific amcha - sensitive binding of human phtlec and phtn3 phages to human plasminogen , an elisa assay , in outline similar to that employed to demonstrate the presence of displayed phlec and phctld giii fusion proteins on the phage particles ( cf . example 2 ), was devised . wells were coated with solutions of human plasminogen ( 10 μg / ml ), with or without addition of 5 mm amcha . control wells were coated with bsa . two identical arrays were established , one was subjected to blocking of excess binding capacity with 3 % skimmed milk , and one was blocked using 3 % skimmed milk supplemented with 5 mm edta . where appropriate , blocking , washing and phage stock solutions were supplemented by 5 mm amcha . the two arrays of wells were incubated with either phtlec -, or phtn3 -, or helper phage samples , and after washing the amount of phage bound in each well was measured using the hrp - conjugated antiphage antibody as above . the results are shown in fig2 , panels a and b , and can be summarized as follows ( a ) in the absence of amcha , binding of human phtlec phages to plasminogen - coated wells generated responses at 8 - 10 times background levels using either formulation of blocking agent , whereas human phtn3 phages generated responses at 4 ( absence of edta ) or 7 ( presence of edta ) times background response levels . ( b ) in the presence of 5 mm amcha , binding of human phtlec - and phtn3 phages to plasminogen was found to be completely abolished . ( c ) phtlec and phtn3 phages showed no binding to bsa , and control helper phages showed no binding to any of the immobilized substances . ( d ) specific binding of human phtlec and phtn3 phages to a specific ligand at moderate binding strength ( about 20 micromolar level ) can be detected with high efficiency at virtually no background using a skimmed - milk blocking agent , well - known in the art of combinatorial phage technology as a preferred agent effecting the reduction of non - specific binding . in conclusion , the results show that the phtlec and phtn3 gene iii fusion proteins displayed on the phage particles exhibit plasminogen - binding properties corresponding to those of authentic tetranectin , and that the physical and biochemical properties of phtlec and phtn3 phages are compatible with their proposed use as vehicles for the generation of combinatorial libraries from which ctld derived units with new binding properties can be selected . all oligonucleotides used in this example were supplied by dna technology ( aarhus , denmark ). the phage library phtlec - lb001 , containing random amino acid residues corresponding to phtlec ( seq id no : 12 ) positions 141 - 146 ( loop 3 ), 150 - 153 ( part of loop 4 )., and residue 168 ( phe in β4 ), was constructed by ligation of 20 μg kpni and muni restricted pphtlec phagemid dna ( cf , example 1 ) with 10 μg of kpni and muni restricted dna fragment amplified from the oligonucleotide htlec - lib1 - tp ( seq id no : 39 ), where n denotes a mixture of 25 % of each of the nucleotides t , c , g , and a , respectively and s denotes a mixture of 50 % of c and g , encoding the appropriately randomized nucleotide sequence and the oligonucleotides htlec - lib1 - rev ( seq id no : 40 ) and htlec - lib1 / 2 - fo ( seq id no : 41 ) as primers using standard conditions . the ligation mixture was used to transform so - called electrocompetent e . coli tg - 1 cells by electroporation using standard procedures . after transformation the e . coli tg - 1 cells were plated on 2 × ty - agar plates containing 0 . 2 mg ampicillin / ml and 2 % glucose and incubated over night at 30 ° c . the phage library phtlec - lb002 , containing random amino acid residues corresponding to phtlec ( seq id no : 12 ) positions 121 - 123 , 125 and 126 ( most of loop 1 ), and residues 150 - 153 ( part of loop 4 ) was constructed by ligation of 20 μg bglii and muni restricted pphtlec phagemid dna ( cf , example 1 ) with 15 μg of bglii and muni restricted dna fragment amplified from the pair of oligonucleotides htlec - lib2 - tprev ( seq id no : 42 ) and htlec - lib2 - tpfo ( seq id no : 43 ), where n denotes a mixture of 25 % of each of the nucleotides t , c , g , and a , respectively and s denotes a mixture of 50 % of c and g , encoding the appropriately randomized nucleotide sequence and the oligonucleotides htlec - lib2 - rev ( seq id no : 44 ) and htlec - lib1 / 2 - fo ( seq id no : 41 ) as primers using standard conditions . the ligation mixture was used to transform so - called electrocompetent e . coli tg - 1 cells by electroporation using standard procedures . after transformation the e . coli tg - 1 cells were plated on 2 × ty - agar plates containing 0 . 2 mg ampicillin / ml and 2 % glucose and incubated overnight at 30 ° c . the titer of the libraries phtlec - lb001 and - lb002 was determined to 1 . 4 * 10 9 and 3 . 2 * 10 9 clones , respectively . six clones from each library were grown and phagemid dna isolated using a standard miniprep procedure , and the nucleotide sequence of the loop - region determined ( dna technology , aarhus , denmark ). one clone from each library failed , for technical reasons , to give reliable nucleotide sequence , and one clone from phtlec - lib001 apparently contained a major deletion . the variation of nucleotide sequences , compared to phtlec ( seq id no : 12 ), of the loop - regions of the other nine clones ( lb001 - 1 , lb001 - 2 , lb001 - 3 , lb001 - 4 , lb002 - 1 , lb002 - 2 , lb002 - 3 , lb002 - 4 , and lb002 - 5 ) is shown in table 3 . the phage library phtctld - lb003 , containing random amino acid residues corresponding to phtctld ( seq id no : 15 ) positions 77 to 7 . 9 and 81 to 82 ( loop 1 ) and 108 to 109 ( loop 4 ) was constructed by ligation of 20 μg bglii and muni restricted pphtctld phagemid dna ( cf . example 1 ) with 10 μg of a bglii and muni restricted dna fragment population encoding the appropriately randomised loop 1 and 4 regions with or without two and three random residue insertions in loop 1 and with three and four random residue insertions in loop 4 . the dna fragment population was amplified , from six so - called assembly reactions combining each of the three loop 1 dna fragments with each of the two loop 4 dna fragments as templates and the oligonucleotides tn - lib3 - rev ( seq id no : 45 ) and loop 3 - 4 - 5 tagfo ( seq id no : 46 ) as primers using standard procedures . each of the three loop 1 fragments was amplified in a reaction with either the oligonucleotides loop1b ( seq id no : 47 ), loop1c ( seq id no : 48 ), or loop1d ( seq id no : 49 ) as template and the oligonucleotides tn - lib3 - rev ( seq id no : 45 ) and tn - kpni - fo ( seq id no : 50 ) as primers , and each of the two dna loop 4 fragments was amplified in a reaction with either the oligonucleotide loop4b ( seq id no : 51 ) or loop4c ( seq id no : 52 ) as template and the oligonucleotides loop3 - 4rev ( seq id no : 53 ) and loop3 - 4fo ( seq id no : 54 ) as primers using standard procedures . in the oligonucleotide sequences n denotes a mixture of 25 % of each of the nucleotides t , c , g , and a , respectively and s denotes a mixture of 50 % of c and g , encoding the appropriately randomized nucleotide sequence . the ligation mixture was used to transform so - called electrocompetent e . coli tg - 1 cells by electroporation using standard procedures . after transformation the e . coli tg - 1 cells were plated on 2 × ty - agar plates containing 0 . 2 mg ampicillin / ml and 2 % glucose and incubated over night at 30 ° c . the size of the resulting library , phtctld - lb003 , was determined to 1 . 4 * 10 10 clones . twenty four clones from the library were grown and phages and phagemid dna isolated . the nucleotide sequences of the loop - regions were determined ( dna technology , aarhus , denmark ) and binding to a polyclonal antibody against tetranectin , anti - tn ( dako a / s , denmark ), analysed in an elisa - type assay using hrp conjugated anti - gene viii ( amersham pharmacia biotech ) as secondary antibody using standard procedures . eighteen clones were found to contain correct loop inserts , one clone contained the wild type loop region sequence , one a major deletion , two contained two or more sequences , and two clones contained a frameshift mutation in the region . thirteen of the 18 clones with correct loop inserts , the wild type clone , and one of the mixed isolates reacted strongly with the polyclonal anti - tn antibody . three of the 18 correct clones reacted weakly with the antibody , whereas , two of the correct clones , the deletion mutant , one of the mixed , and the two frameshift mutants did not show a signal above background . approximately 10 11 phages from the phtctld - lb003 library was used for selection in two rounds on the polyclonal anti - tn antibody by panning in maxisorb immunotubes ( nunc , denmark ) using standard procedures . fifteen clones out of 7 * 10 7 from the plating after the second selection round were grown and phagemid dna isolated and the nucleotide sequence determined . all 15 clones were found to encode correct and different loop sequences . in order to demonstrate that tetranectin derived ctld bearing phages can be selected from a population of phages , mixtures of phtctld phages isolated from a e . coli tg1 culture transformed with the phagemid pphtctld ( cf , example 1 ) after infection with m13k07 helper phage and phages isolated from a culture transformed with the phagemid pphtcpb after infection with m13k07 helper phage at ratios of 1 : 10 and 1 : 10 5 , respectively were used in a selection experiment using panning in 96 - well maxisorb micro - titerplates ( nunc , denmark ) and with human plasminogen as antigen . the pphtcpb phagemid was constructed by ligation of the double stranded oligonucleotide ( seq id no : 55 ) with the appropriate restriction enzyme overhang sequences into kpni and muni restricted pphtctld phagemid dna . the pphtcbp phages derived upon infection with the helper phages displays only the wild type m13 gene iii protein because of the translation termination codons introduced into the ctld coding region of the resulting pphtcpb phagemid ( seq id no : 56 ). the selection experiments were performed in 96 well micro titer plates using standard procedures . briefly , in each well 3 μg of human plasminogen in 100 μl pbs ( pbs , 0 . 2 g kcl , 0 . 2 g kh 2 po 4 , 8 g nacl , 1 . 44 g na 2 hpo 4 , 2h 2 o , water to 1 l , and adjusted to ph 7 . 4 with naoh ) or 100 μl pbs ( for analysis of non specific binding ) was used for over night coating at 4 ° c . and at 37 ° c . for one hour . after washing once with pbs , wells were blocked with 400 μl pbs and 3 % non fat dried milk for one hour at 37 ° c . after blocking wells were washed once in pbs and 0 . 1 % tween 20 and three times with pbs before the addition of phages suspended in 100 μl pbs , 3 % non fat dried milk . the phages were allowed to bind at 37 ° c . for one hour before washing three times with pbs , tween 20 and three times with pbs . bound phages were eluted from each well by trypsin digestion in 100 μl ( 1 mg / ml trypsin in pbs ) for 30 min . at room temperature , and used for infection of exponentially growing e . coli tg1 cells before plating and titration on 2 × ty agar plates containing 2 % glucose and 0 . 1 mg / ml ampicillin . initially ( round 1 ), 10 12 phtctld phages ( a series ), a mixture of 10 phtctld phages and 10 11 phtcpb phages ( b series ), or a mixture of 10 6 phtctld and 10 11 phtcpb phages ( c series ) were used . in the following round ( round 2 ) 10 11 phages of the output from each series were used . results from the two rounds of selection are summarised in table 4 . phagemid dna from 12 colonies from the second round of plating together with 5 colonies from a plating of the initial phage mixtures was isolated and the nucleotide sequence of the ctld region determined . from the initial 1 / 10 mixture ( b series ) of phtctld / phtcpb one out of five were identified as the ctld sequence . from the initial 1 / 10 5 mixture ( c series ) all five sequences were derived from the pphtcpb phagemid . after round 2 nine of the twelve sequences analysed from the b series and all twelve sequences from the c series were derived from the pphtctld phagemid . in order to demonstrate that tetranectin derived ctld - bearing phages can be selected from a population of phages , mixtures of phtctld phages isolated from a e . coli tg1 culture transformed with the phagemid pphtctld ( cf , example 1 ) after infection with m13k07 helper phage and phages isolated from a culture transformed with the phagemid pphtcpb ( cf , example 6 ) after infection with m13k07 helper phage at ratios of 1 : 10 2 and 1 : 10 6 , respectively were used in a selection experiment using panning in 96 - well maxisorb micro - titerplates ( nunc , denmark ) and with human plasminogen as antigen using standard procedures . briefly , in each well 3 μg of human plasminogen in 100 μl pbs ( pbs , 0 . 2 g kcl , 0 . 2 g kh 2 po 4 , 8 g nacl , 1 . 44 g na 2 hpo 4 , 2h 2 o , water to 1 l , and adjusted to ph 7 . 4 with naoh ) or 100 μl pbs ( for analysis of non specific binding ) was used for over night coating at 4 ° c . and at 37 ° c . for one hour . after washing once with pbs , wells were blocked with 400 μl pbs and 3 % non fat dried milk for one hour at 37 ° c . after blocking wells were washed once in pbs and 0 . 1 % tween 20 and three times with pbs before the addition of phages suspended in 100 μl pbs , 3 % non fat dried milk . the phages were allowed to bind at 37 ° c . for one hour before washing 15 times with pbs , tween 20 , and 15 times with pbs . bound phages were eluted from each well by 100 μl 0 . 1 m triethyl - amine for 10 min at room temperature , and upon neutralisation with 0 . 5 vol . 1 m tris - hcl ph 7 . 4 , used for infection of exponentially growing e . coli tg1 cells before plating and titration on 2 × ty agar plates containing 2 % glucose and 0 . 1 mg / ml ampicillin . initially ( round 1 ) 10 12 phtctld phages ( a series ), a mixture of 10 9 phtctld phages and 10 11 phtcpb phages ( b series ), or a mixture of 10 5 phtctld and 10 11 phtcpb phages ( c series ) were used . in the following round ( round 2 ) 10 11 phages of the output from each series were used . results from the two rounds of selection are summarised in table 5 . phage mixtures from the a and the b series from the second round of selection were grown using a standard procedure , and analysed for binding to plasminogen in an elisa - type assay . briefly , in each well 31 g of plasminogen in 100 μl pbs ( pbs , 0 . 2 g kcl , 0 . 2 g kh 2 po 4 , 8 g nacl , 1 . 44 g na 2 hpo 4 2h 2 o water to 1 l , and adjusted to ph 7 . 4 with naoh ) or 100 μl pbs ( for analysis of non specific binding ) was used for over night coating at 4 ° c . and at 37 ° c . for one hour . after washing once with pbs , wells were blocked with 400 μl pbs and 3 % non fat dried milk for one hour at 37 ° c . after blocking wells were washed once in pbs and 0 . 1 % tween 20 and three times with pbs before the addition of phages suspended in 100 μl pbs , 3 % non fat dried milk . the phage mixtures were allowed to bind at 37 ° c . for one hour before washing three times with pbs , tween 20 , and three times with pbs . after washing , 50 μl of a 1 : 5000 dilution of a hrp - conjugated anti - gene viii antibody ( amersham pharmacia biotech ) in pbs , 3 % non fat dried milk was added to each well and incubated at 37 ° c . for one hour . after binding of the “ secondary ” antibody wells were washed three times with pbs , tween 20 , and three times with pbs before the addition of 50 μl of tmb substrate ( dako - tmb one - step substrate system , code : s1600 , dako , denmark ). reaction was allowed to proceed for 20 min . before quenching with 0 . 5 vol . 0 . 5 m h 2 so 4 , and analysis . the result of the elisa analysis confirmed specific binding to plasminogen of phages in both series ( fig2 ). selection of phases from the library phtlec - lb002 binding to hen egg white lysozyme . 1 . 2 * 10 12 phages , approximately 250 times the size of the original library , derived from the phtlec - lb002 library ( cf , example 4 ) were used in an experimental procedure for the selection of phages binding to hen egg white lysozyme involving sequential rounds of panning using standard procedures . briefly , 30 μg of hen egg white lysozyme in 1 ml pbs ( pbs , 0 . 2 g kcl , 0 . 2 g kh 2 po 4 , 8 g nacl , 1 . 44 g na 2 hpo 4 , 2h 2 o , water to 1 l , and adjusted to ph 7 . 4 with naoh ) or 1 ml pbs ( for analysis of non specific binding ) was used for over night coating of maxisorb immunotubes ( nunc , denmark ) at 4 ° c . and at 37 ° c . for one hour . after washing once with pbs , tubes were filled and blocked with pbs and 3 % non fat dried milk for one hour at 37 ° c . after blocking tubes were washed once in pbs , 0 . 1 % tween 20 and three times with pbs before the addition of phages suspended in 1 ml pbs , 3 % non fat dried milk . the phages were allowed to bind at 37 ° c . for one hour before washing six times with pbs , tween 20 and six times with pbs . bound phages were eluted from each well by 1 ml 0 . 1 m triethylamine for 10 min at room temperature , and upon neutralisation with 1 m tris - hcl ph 7 . 4 , used for infection of exponentially growing e . coli tg1 cells before plating and titration on 2 × ty agar plates containing 2 % glucose and 0 . 1 mg / ml ampicillin . in the subsequent rounds of selection approximately 10 12 phages derived from a culture grown from the colonies plated after infection with the phages eluted from the lysozyme coated tube were used in the panning procedure . however , the stringency in binding was increased by increasing the number of washing step after phage panning from six to ten . the results from the selection procedure is shown in table 7 . phages were grown from twelve clones isolated from the third round of selection in order to analyse the specificity of binding using a standard procedure , and analysed for binding to hen egg white lysozyme and human β 2 - microglobulin in an elisa - type assay . briefly , in each well 3 μg of hen egg white lysozyme in 100 μl pbs ( pbs , 0 . 2 g kcl , 0 . 2 g kh 2 po 4 , 8 g nacl , 1 . 44 g na 2 hpo 4 , 2h 2 o , water to 1 l , and adjusted to ph 7 . 4 with naoh ), or 3 μg of human β 2 - microglobulin , or 100 μl pbs ( for analysis of non specific binding ) was used for over night coating at 4 ° c . and at 37 ° c . for one hour . after washing once with pbs , wells were blocked with 400 μl pbs and 3 % non fat dried milk for one hour at 37 ° c . after blocking wells were washed once in pbs and 0 . 1 % tween 20 and three times with pbs before the addition of phages suspended in 100 μl pbs , 3 % non fat dried milk . the phages were allowed to bind at 37 ° c . for one hour before washing three times with pbs , tween 20 and three times with pbs . after washing , 50 μl of a 1 to 5000 dilution of a hrp - conjugated anti - gene viii antibody ( amersham pharmacia biotech ) in pbs , 3 % non fat dried milk was added to each well and incubated at 37 ° c . for one hour . after binding of the “ secondary ” antibody wells were washed three times with pbs , tween 20 and three times with pbs before the addition of 50 μl of tmb substrate ( dako - tmb one - step substrate system , code : s1600 , dako , denmark ). reaction was allowed to proceed for 20 min before quenching with 0 . 5 m h 2 so 4 . results showing relatively weak but specific binding to lysozyme are summarised in fig2 . construction of the rat mannose - binding protein ctld ( rmbp ) derived phagemid ( pprmbp ) and human lung surfactant protein d ctld ( h - sp - d ) derived phagemid ( pphsp - d ) the phagemid , pprmbp , is constructed by ligation of the sfi i and not i restricted dna fragment amplified from cdna , isolated from rat liver ( drickamer , k ., et al ., j . biol . chem . 1987 , 262 ( 6 ): 2582 - 2589 ) ( with the oligonucleotide primers sfimbp 5 - cggctgagcggcccagccggccatggccgagccaaacaagttgcatgccttctcc - 3 ′ [ seq id no : 62 ] and notmbp 5 ′- gcactcctgcggccgcggctgggaactcgcagac - 3 ′ [ seq id no : 63 ]) into a sfi i and not i precut vector , pcantab 5e supplied by amersham pharmacia biotech ( code no . 27 - 9401 - 01 ) using standard procedures . outlines of the resulting pprmbp is shown in fig3 and the nucleotide sequence of prmbp is given as ( seq id no : 58 ). the amino acid sequence encoded by the prmbp insert is shown in fig3 ( seq id no : 59 ). the phagemid , pphsp - d , is constructed by ligation of the sfi i and not i restricted dna fragment amplified from cdna , isolated from human lung ( lu , j ., et al ., biochem j . 1992 jun 15 ; 284 : 795 - 802 ) ( with the oligonucleotide primers sfisp - d 5 ′- cggctgagcggcccagccggccatggccgagccaaagaaagttgagctcttccc - 3 ′ [ seq id no : 64 ] and notsp - d 5 ′- gcactcctgcggccgcgaactcgcagaccacaagac - 3 ′ [ seq id no : 65 ]) into a sfi i and not i precut vector , pcantab 5e supplied by amersham pharmacia biotech ( code no . 27 - 9401 - 01 ) using standard procedures . outlines of the resulting pphsp - d is shown in fig3 and the nucleotide sequence of phsp - d , is given as ( seq id no : 60 ). the amino acid sequences encoded by the phsp - d insert is shown in fig3 ( seq id no : 61 ). the phage library prmbp - lb001 , containing random amino acid residues corresponding to prmbp ctld ( seq id no : 59 ) positions 71 to 73 or 70 to 76 ( loop 1 ) and 97 to 101 or 100 to 101 ( loop 4 ) is constructed by ligation of 20 μg sfii and noti restricted pprmbp phagemid dna ( cf . example 10 ) with 10 μg of a sfii and noti restricted dna fragment population encoding the appropriately randomised loop 1 and 4 regions . the dna fragment population is amplified , from nine assembly reactions combining each of the three loop 1 dna fragments with each of the three loop 4 dna fragments as templates and the oligonucleotides sfi - tag 5 ′- cggctgagcggcccagc - 3 ′ ( seq id no : 74 ) and not - tag 5 ′- gcactcctgcggccgcg - 3 ′ ( seq id no : 75 ) as primers using standard procedures . each of the three loop 1 fragments is amplified in a primary pcr reaction with pprmbp phagmid dna ( cf . example 10 ) as template and the oligonucleotides mbploop1a fo ( seq id no : 66 ), mbploop1b fo ( seq id no : 67 ) or mbploop1c fo ( seq id no : 68 ) and sfimbp ( seq id no : 62 ) as primers , and further amplified in a secondary pcr reaction using sfi - tag ( seq id no : 74 ) and mbploop1 - tag fo ( seq id no : 69 ). each of the three dna loop 4 fragments is amplified in a primary pcr reaction with pprmbp phagemid dna ( cf . example 10 ) as template and the oligonucleotides mbploop4a rev ( seq id no : 71 ), mbploop4b rev ( seq id no : 72 ) or mbploop4c rev ( seq id no : 73 ) and notmbp ( seq id no : 63 ) as primers using standard procedures and further amplified in a secondary pcr reaction using mbploop4 - tag rev ( seq id no : 70 ) and not - tag ( seq id no : 63 ). in the oligonucleotide sequences n denotes a mixture of 25 % of each of the nucleotides t , c , g , and a , respectively , and s denotes a mixture of 50 % of c and g , encoding the appropriately randomized nucleotide sequence . the ligation mixture is used to transform so - called electrocompetent e . coli tg - 1 cells by electroporation using standard procedures . after transformation the e . coli tg - 1 cells are plated on 2 × ty - agar plates containing 0 . 2 mg ampicillin / ml and 2 % glucose and incubated over night at 30 ° c . the phage library phsp - d - lb001 , containing random amino acid residues corresponding to phsp - d ctld insert ( seq id no : 61 ) positions 74 to 76 or 73 to 79 ( loop 1 ) and 100 to 104 or 103 to 104 ( loop 4 ) is constructed by ligation of 20 μg sfii and noti restricted pphsp - d phagemid dna ( cf . example 10 ) with 10 μg of a sfii and noti restricted dna fragment population encoding the appropriately randomised loop 1 and 4 regions . the dna fragment population is amplified , from nine assembly reactions combining each of the three loop 1 dna fragments with each of the three loop 4 dna fragments as templates and the oligonucleotides sfi - tag 5 ′- cggctgagcggcccagc - 3 ′ ( seq id no : 74 ) and not - tag 5 ′- gcactcctgcggccgcg - 3 ′ ( seq id no : 75 ) as primers using standard procedures . each of the three loop 1 fragments is amplified in a primary pcr reaction with pphsp - d phagemid dna ( cf . example 10 ) as template and the oligonucleotides spdloop1a fo ( seq id no : 76 ), sp - dloop1b fo ( seq id no : 77 ) or sp - dloop1c fo ( seq id no : 78 ) and sfisp - d ( seq id no : 64 ) as primers , and further amplified in a pcr reaction using sfi - tag ( seq id no : 74 ) and sp - dloop1 - tag fo ( seq id no : 79 ) as primers . each of the three dna loop 4 fragments is amplified in a primary pcr reaction with pphsp - d phagemid dna ( cf . example 10 ) as template and the oligonucleotides sp - dloop4a rev ( seq id no : 81 ), sp - dloop4b rev ( seq id no : 82 ) or sp - dloop4c rev ( seq id no : 83 ) and notsp - d ( seq id no : 65 ) as primers using standard procedures and further amplified in a pcr reaction using sp - dloop4 - tag rev ( seq id no : 80 ) and not - tag ( seq id no : 75 ) as primers . in the oligonucleotide sequences n denotes a mixture of 25 % of each of the nucleotides t , c , g , and a , respectively , and s denotes a mixture of 50 % of c and g , encoding the appropriately randomized nucleotide sequence . the ligation mixture is used to transform so - called electrocompetent e . coli tg - 1 cells by electroporation using standard procedures . after transformation the e . coli tg - 1 cells are plated on 2 × ty - agar plates containing 0 . 2 mg ampicillin / ml and 2 % glucose and incubated over night at 30 ° c . all oligonucleotides used in this example were supplied by dna technology ( aarhus , denmark ). the phage library phtctld - lb004 , containing random amino acid residues corresponding to phtctld ( seq id no : 15 ) positions 97 to 102 or 98 to 101 ( loop 3 ) and positions 116 to 122 or 118 to 120 ( loop 5 ) was constructed by ligation of 20 μg kpni and muni restricted pphtctld phagemid dna ( cf . example 1 ) with 10 μg of a kpni and muni restricted dna fragment population encoding the randomised loop 3 and 5 regions . the dna fragment population was amplified from nine primary pcr reactions combining each of the three loop 3 dna fragments with each of the three loop 5 dna fragments . the fragments was amplified with either of the oligonucleotides loop3a ( seq id no : 84 ), loop3b ( seq id no : 85 ), or loop3c ( seq id no : 86 ) as template and loop5a ( seq id no : 87 ), loop5b ( seq id no : 88 ) or loop5c ( seq id no : 89 ) and loop3 - 4rev ( seq id no : 91 ) as primers . the dna fragments were further amplified in pcr reactions , using the primary pcr product as template and the oligonucleotide loop3 - 4rev ( seq id no : 91 ) and loop3 - 4 - 5tag fo ( seq id no : 90 ) as primers . all pcr reactions were performed using standard procedures . in the oligonucleotide sequences n denotes a mixture of 25 % of each of the nucleotides t , c , g , and a , respectively and s denotes a mixture of 50 % of c and g , encoding the appropriately randomised nucleotide sequence . the ligation mixture was used to transform so - called electrocompetent e . coli tg - 1 cells by electroporation using standard procedures . after transformation the e . coli tg - 1 cells were plated on 2 × ty - agar plates containing 0 . 2 mg ampicillin / ml and 2 % glucose and incubated over night at 30 ° c . the size of the resulting library , phtctld - lb004 , was determined to 7 * 109 clones . sixteen clones from the library were picked and phagemid dna isolated . the nucleotide sequence of the loop - regions were determined ( dna technology , aarhus , denmark ). thirteen clones were found to contain correct loop inserts and three clones contained a frameshift mutation in the region . selection of phtlec - phages and phtctld - phages binding to the blood group a sugar moiety immobilised on human serum albumin phages grown from glycerol stocks of the libraries phtlec - lb001 and phtlec - lb002 ( cf . example 4 ) and phages grown from a glycerol stock of the library phtctld - lb003 ( cf . example 5 ), using a standard procedure , were used in an experiment designed for the selection of phtlec - and phtctld derived phages with specific affinity to the blood group a sugar moiety immobilized on human serum albumin ; a - ha , by panning in 96 - well maxisorb micro - titerplates ( nunc , denmark ) using standard procedures . initially , the phage supernatants were precipitated with 0 . 3 vol . of a solution of 20 % polyethylene glycol 6000 ( peg ) and 2 . 5 m nacl , and the pellets re - suspended in te - buffer ( 10 mm tris - hcl ph 8 , 1 mm edta ). after titration on e . coli tg - 1 cells , phages derived from phtlec - lb001 and - lb002 were mixed (# 1 ) in a . 1 : 1 ratio and adjusted to 5 * 1012 pfu / ml in 2 * ty medium , and phages grown from the phtctld - lb003 library (# 4 ) were adjusted to 2 . 5 * 1012 pfu / ml in 2 * ty medium . one microgram of the “ antigen ”, human blood group a trisaccharide immobilised on human serum albumin , a - ha , ( glycorex ab , lund , sweden ) in 100 μl pbs ( pbs , 0 . 2 g kcl , 0 . 2 g kh 2 po 4 , 8 g nacl , 1 . 44 g na 2 hpo 4 , 2h 2 o , water to 1 l , and adjusted to ph 7 . 4 with naoh ), in each of three wells , was coated over night at 4 ° c . and at room temperature for one hour , before the first round of panning . after washing once with pbs , wells were blocked with 300 μl pbs and 3 % non fat dried milk for one hour at room temperature . after blocking wells were washed once in pbs and 0 . 1 % tween 20 and three times with pbs before the addition of a mixture of 50 μl of the phage suspension and 50 μl pbs , 6 % non fat dried milk . the phages were allowed to bind at room temperature for two hours before washing eight times with pbs , tween 20 , and eight times with pbs . bound phages were eluted from each well by trypsin digestion in 100 μl ( 1 mg / ml trypsin in pbs ) for 30 min . at room temperature , and used for infection of exponentially growing e . coli tg1 cells before plating and titration on 2 × ty agar plates containing 2 % glucose and 0 . 1 mg / ml ampicillin . in the second round of selection , 150 μl of crude phage supernatant , grown from the first round output colonies , was mixed with 150 μl pbs , 6 % non fat dried milk , and used for panning distributing 100 μl of the mixture in each of three a - ha coated wells , as previously described . stringency in binding was increased by increasing the number of washing steps from 16 to 32 . 300 μl of phage mixture was also used for panning in three wells , which had received no antigen as control . in the third round of selection , 150 μl of crude phage supernatant , grown from the second round output colonies , was mixed with 150 μl pbs , 6 % non fat dried milk , and used for panning distributing 100 μl of the mixture in each of three a - ha coated wells , as previously described . the number of washing steps was again 32 . 300 μl of phage mixture was also used for panning in three wells , which had received no antigen as control . the results from the selection procedure are summarised in table 8 48 clones from each of the # 1 and # 4 series were picked and grown in a 96 well microtiter tray and phages produced by infection with m13k07 helper phage using a standard procedure . phages from the 96 phage supernatants were analysed for binding to the a - ha antigen and for non - specific binding to hen egg white lysozyme using an elisa - type assay . briefly , in each well 1 μg of a - ha in 100 μl pbs ( pbs , 0 . 2 g kcl , 0 . 2 g kh 2 po 4 , 8 g nacl , 1 . 44 g na 2 hpo 4 , 2h 2 o , water to 1 l , and adjusted to ph 7 . 4 with naoh ) or 1 μg of hen egg white lysozyme in 100 μl pbs ( for analysis of non specific binding ) was used for over night coating at 4 ° c . and at room temperature for one hour . after washing once with pbs , wells were blocked with 300 μl pbs and 3 % non fat dried milk for one hour at room temperature . after blocking wells were washed once in pbs and 0 . 1 % tween 20 and three times with pbs before the addition of 50 μl phage supernatant in 50 μl pbs , 6 % non fat dried milk . the phage mixtures were allowed to bind at room temperature for two hours before washing three times with pbs , tween 20 , and three times with pbs . after washing , 50 μl of a 1 : 5000 dilution of a hrp - conjugated anti - gene viii antibody ( amersham pharmacia biotech ) in pbs , 3 % non fat dried milk , was added to each well and incubated at room temperature for one hour . after binding of the “ secondary ” antibody wells were washed three times with pbs , tween 20 , and three times with pbs before the addition of 50 μl of tmb substrate ( dako - 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( 2000 ). “ cloning , mapping and genomic organization of a fish c - type lectin gene from homozygous clones of rainbow trout ( oncorhynchos mykiss )”. biochim . et biophys . acta 1494 : 14 - 22 gga tcc atc gag ggt agg ggc gag cca cca acc cag aag ccc aag aag 48 att gta aat gcc aag aaa gat gtt gtg aac aca aag atg ttt gag gag 96 ctc aag agc cgt ctg gac acc ctg gcc cag gag gtg gcc ctg ctg aag 144 gag cag cag gcc ctg cag acg gtc gtc ctg aag ggg acc aag gtg cac 192 atg aaa gtc ttt ctg gcc ttc acc cag acg aag acc ttc cac gag gcc 240 agc gag gac tgc atc tcg cgc ggg ggc acc ctg agc acc cct cag act 288 ser glu asp cys ile ser arg gly gly thr leu ser thr pro gln thr ggc tcg gag aac gac gcc ctg tat gag tac ctg cgc cag agc gtg ggc 336 gly ser glu asn asp ala leu tyr glu tyr leu arg gln ser val gly aac gag gcc gag atc tgg ctg ggc ctc aac gac atg gcg gcc gag ggc 384 acc tgg gtg gac atg acc ggt acc cgc atc gcc tac aag aac tgg gag 432 thr trp val asp met thr gly thr arg ile ala tyr lys asn trp glu act gag atc acc gcg caa ccc gat ggc ggc aag acc gag aac tgc gcg 480 thr glu ile thr ala gln pro asp gly gly lys thr glu asn cys ala gtc ctg tca ggc gcg gcc aac ggc aag tgg ttc gac aag cgc tgc cgc 528 val leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg gat caa ttg ccc tac atc tgc cag ttc ggg atc gtg taagctt 571 ser glu asp cys ile ser arg gly gly thr leu ser thr pro gln thr gly ser glu asn asp ala leu tyr glu tyr leu arg gln ser val gly thr trp val asp met thr gly thr arg ile ala tyr lys asn trp glu thr glu ile thr ala gln pro asp gly gly lys thr glu asn cys ala val leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg gga tcc atc gag ggt agg gcc ctg cag acg gtc gtc ctg aag ggg acc 48 gly ser ile glu gly arg ala leu gln thr val val leu lys gly thr aag gtg cac atg aaa gtc ttt ctg gcc ttc acc cag acg aag acc ttc 96 cac gag gcc agc gag gac tgc atc tcg cgc ggg ggc acc ctg agc acc 144 his glu ala ser glu asp cys ile ser arg gly gly thr leu ser thr cct cag act ggc tcg gag aac gac gcc ctg tat gag tac ctg cgc cag 192 pro gln thr gly ser glu asn asp ala leu tyr glu tyr leu arg gln agc gtg ggc aac gag gcc gag atc tgg ctg ggc ctc aac gac atg gcg 240 ser val gly asn glu ala glu ile trp leu gly leu asn asp met ala gcc gag ggc acc tgg gtg gac atg acc ggt acc cgc atc gcc tac aag 288 ala glu gly thr trp val asp met thr gly thr arg ile ala tyr lys aac tgg gag act gag atc acc gcg caa ccc gat ggc ggc aag acc gag 336 asn trp glu thr glu ile thr ala gln pro asp gly gly lys thr glu aac tgc gcg gtc ctg tca ggc gcg gcc aac ggc aag tgg ttc gac aag 384 asn cys ala val leu ser gly ala ala asn gly lys trp phe asp lys cgc tgc cgc gat caa ttg ccc tac atc tgc cag ttc ggg atc gtg 429 arg cys arg asp gln leu pro tyr ile cys gln phe gly ile val gly ser ile glu gly arg ala leu gln thr val val leu lys gly thr his glu ala ser glu asp cys ile ser arg gly gly thr leu ser thr pro gln thr gly ser glu asn asp ala leu tyr glu tyr leu arg gln ser val gly asn glu ala glu ile trp leu gly leu asn asp met ala ala glu gly thr trp val asp met thr gly thr arg ile ala tyr lys asn trp glu thr glu ile thr ala gln pro asp gly gly lys thr glu asn cys ala val leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp gln leu pro tyr ile cys gln phe gly ile val ggcccag ccg gcc atg gcc gag cca cca acc cag aag ccc aag aag att 49 gta aat gcc aag aaa gat gtt gtg aac aca aag atg ttt gag gag ctc 97 aag agc cgt ctg gac acc ctg gcc cag gag gtg gcc ctg ctg aag gag 145 cag cag gcc ctg cag acg gtc tgc ctg aag ggg acc aag gtg cac atg 193 aaa tgc ttt ctg gcc ttc acc cag acg aag acc ttc cac gag gcc agc 241 gag gac tgc atc tcg cgc ggg ggc acc ctg agc acc cct cag act ggc 289 glu asp cys ile ser arg gly gly thr leu ser thr pro gln thr gly tcg gag aac gac gcc ctg tat gag tac ctg cgc cag agc gtg ggc aac 337 ser glu asn asp ala leu tyr glu tyr leu arg gln ser val gly asn gag gcc gag atc tgg ctg ggc ctc aac gac atg gcg gcc gag ggc acc 385 tgg gtg gac atg acc ggc gcc cgc atc gcc tac aag aac tgg gag act 433 trp val asp met thr gly ala arg ile ala tyr lys asn trp glu thr gag atc acc gcg caa ccc gat ggc ggc aag acc gag aac tgc gcg gtc 481 glu ile thr ala gln pro asp gly gly lys thr glu asn cys ala val ctg tca ggc gcg gcc aac ggc aag tgg ttc gac aag cgc tgc cgc gat 529 leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp cag ctg ccc tac atc tgc cag ttc ggg atc gtg gcg gccgc 570 ala lys lys asp val val asn thr lys met phe glu glu leu lys ser asn asp ala leu tyr glu tyr leu arg gln ser val gly asn glu ala glu ile trp leu gly leu asn asp met ala ala glu gly thr trp val asp met thr gly ala arg ile ala tyr lys asn trp glu thr glu ile thr ala gln pro asp gly gly lys thr glu asn cys ala val leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp gln leu ggcccag ccg gcc atg gcc gcc ctg cag acg gtc tgc ctg aag ggg acc 49 aag gtg cac atg aaa tgc ttt ctg gcc ttc acc cag acg aag acc ttc 97 cac gag gcc agc gag gac tgc atc tcg cgc ggg ggc acc ctg agc acc 145 his glu ala ser glu asp cys ile ser arg gly gly thr leu ser thr cct cag act ggc tcg gag aac gac gcc ctg tat gag tac ctg cgc cag 193 pro gln thr gly ser glu asn asp ala leu tyr glu tyr leu arg gln agc gtg ggc aac gag gcc gag atc tgg ctg ggc ctc aac gac atg gcg 241 ser val gly asn glu ala glu ile trp leu gly leu asn asp met ala gcc gag ggc acc tgg gtg gac atg acc ggc gcc cgc atc gcc tac aag 289 ala glu gly thr trp val asp met thr gly ala arg ile ala tyr lys aac tgg gag act gag atc acc gcg caa ccc gat ggc ggc aag acc gag 337 asn trp glu thr glu ile thr ala gln pro asp gly gly lys thr glu aac tgc gcg gtc ctg tca ggc gcg gcc aac ggc aag tgg ttc gac aag 385 asn cys ala val leu ser gly ala ala asn gly lys trp phe asp lys cgc tgc cgc gat cag ctg ccc tac atc tgc cag ttc ggg atc gtg gcg 433 arg cys arg asp gln leu pro tyr ile cys gln phe gly ile val ala ala ser glu asp cys ile ser arg gly gly thr leu ser thr pro gln thr gly ser glu asn asp ala leu tyr glu tyr leu arg gln ser val gly thr trp val asp met thr gly ala arg ile ala tyr lys asn trp glu thr glu ile thr ala gln pro asp gly gly lys thr glu asn cys ala val leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp gln leu pro tyr ile cys gln phe gly ile val ala ggcccag ccg gcc atg gcc gag cca cca acc cag aag ccc aag aag att 49 gta aat gcc aag aaa gat gtt gtg aac aca aag atg ttt gag gag ctc 97 aag agc cgt ctg gac acc ctg gcc cag gag gtg gcc ctg ctg aag gag 145 cag cag gcc ctg cag acg gtc gtc ctg aag ggg acc aag gtg cac atg 193 aaa gtc ttt ctg gcc ttc acc cag acg aag acc ttc cac gag gcc agc 241 gag gac tgc atc tcg cgc ggg ggc acc ctg agc acc cct cag act ggc 289 glu asp cys ile ser arg gly gly thr leu ser thr pro gln thr gly tcg gag aac gac gcc ctg tat gag tac ctg cgc cag agc gtg ggc aac 337 ser glu asn asp ala leu tyr glu tyr leu arg gln ser val gly asn gag gcc gag atc tgg ctg ggc ctc aac gac atg gcg gcc gag ggc acc 385 tgg gtg gac atg acc ggt acc cgc atc gcc tac aag aac tgg gag act 433 trp val asp met thr gly thr arg ile ala tyr lys asn trp glu thr gag atc acc gcg caa ccc gat ggc ggc aag acc gag aac tgc gcg gtc 481 glu ile thr ala gln pro asp gly gly lys thr glu asn cys ala val ctg tca ggc gcg gcc aac ggc aag tgg ttc gac aag cgc tgc cgc gat 529 leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp caa ttg ccc tac atc tgc cag ttc ggg atc gtg gcg gccgc 570 ala lys lys asp val val asn thr lys met phe glu glu leu lys ser asn asp ala leu tyr glu tyr leu arg gln ser val gly asn glu ala glu ile trp leu gly leu asn asp met ala ala glu gly thr trp val asp met thr gly thr arg ile ala tyr lys asn trp glu thr glu ile thr ala gln pro asp gly gly lys thr glu asn cys ala val leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp gln leu ggcccag ccg gcc atg gcc gcc ctg cag acg gtc gtc ctg aag ggg acc 49 aag gtg cac atg aaa gtc ttt ctg gcc ttc acc cag acg aag acc ttc 97 cac gag gcc agc gag gac tgc atc tcg cgc ggg ggc acc ctg agc acc 145 his glu ala ser glu asp cys ile ser arg gly gly thr leu ser thr cct cag act ggc tcg gag aac gac gcc ctg tat gag tac ctg cgc cag 193 pro gln thr gly ser glu asn asp ala leu tyr glu tyr leu arg gln agc gtg ggc aac gag gcc gag atc tgg ctg ggc ctc aac gac atg gcg 241 ser val gly asn glu ala glu ile trp leu gly leu asn asp met ala gcc gag ggc acc tgg gtg gac atg acc ggt acc cgc atc gcc tac aag 289 ala glu gly thr trp val asp met thr gly thr arg ile ala tyr lys aac tgg gag act gag atc acc gcg caa ccc gat ggc ggc aag acc gag 337 asn trp glu thr glu ile thr ala gln pro asp gly gly lys thr glu aac tgc gcg gtc ctg tca ggc gcg gcc aac ggc aag tgg ttc gac aag 385 asn cys ala val leu ser gly ala ala asn gly lys trp phe asp lys cgc tgc cgc gat caa ttg ccc tac atc tgc cag ttc ggg atc gtg gcg 433 arg cys arg asp gln leu pro tyr ile cys gln phe gly ile val ala ala ser glu asp cys ile ser arg gly gly thr leu ser thr pro gln thr gly ser glu asn asp ala leu tyr glu tyr leu arg gln ser val gly thr trp val asp met thr gly thr arg ile ala tyr lys asn trp glu thr glu ile thr ala gln pro asp gly gly lys thr glu asn cys ala val leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp gln leu pro tyr ile cys gln phe gly ile val ala gga tcc atc cag ggt cgc gag tca ccc act ccc aag gcc aag aag gct 48 gca aat gcc aag aaa gat ttg gtg agc tca aag atg ttc gag gag ctc 96 aag aac agg atg gat gtc ctg gcc cag gag gtg gcc ctg ctg aag gag 144 aag cag gcc tta cag act gtg gtc ctg aag ggc acc aag gtg aac ttg 192 aag gtc ctc ctg gcc ttc acc caa ccg aag acc ttc cat gag gcg agc 240 lys val leu leu ala phe thr gln pro lys thr phe his glu ala ser gag gac tgc atc tcg caa ggg ggc acg ctg ggc acc ccg cag tca gag 288 cta gag aac gag gcg ctg ttc gag tac gcg cgc cac agc gtg ggc aac 336 leu glu asn glu ala leu phe glu tyr ala arg his ser val gly asn gat gcg gag atc tgg ctg ggc ctc aac gac atg gcc gcg gaa ggc gcc 384 tgg gtg gac atg acc ggt acc ctc ctg gcc tac aag aac tgg gag acg 432 trp val asp met thr gly thr leu leu ala tyr lys asn trp glu thr gag atc acg acg caa ccc gac ggc ggc aaa gcc gag aac tgc gcc gcc 480 glu ile thr thr gln pro asp gly gly lys ala glu asn cys ala ala ctg tct ggc gca gcc aac ggc aag tgg ttc gac aag cga tgc cgc gat 528 leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp caa ttg ccc tac atc tgc cag ttt gcc att gtg taagctt 568 lys val leu leu ala phe thr gln pro lys thr phe his glu ala ser leu glu asn glu ala leu phe glu tyr ala arg his ser val gly asn trp val asp met thr gly thr leu leu ala tyr lys asn trp glu thr glu ile thr thr gln pro asp gly gly lys ala glu asn cys ala ala leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp gga tcc atc cag ggt cgc gcc tta cag act gtg gtc ctg aag ggc acc 48 aag gtg aac ttg aag gtc ctc ctg gcc ttc acc caa ccg aag acc ttc 96 cat gag gcg agc gag gac tgc atc tcg caa ggg ggc acg ctg ggc acc 144 his glu ala ser glu asp cys ile ser gln gly gly thr leu gly thr ccg cag tca gag cta gag aac gag gcg ctg ttc gag tac gcg cgc cac 192 pro gln ser glu leu glu asn glu ala leu phe glu tyr ala arg his agc gtg ggc aac gat gcg gag atc tgg ctg ggc ctc aac gac atg gcc 240 ser val gly asn asp ala glu ile trp leu gly leu asn asp met ala gcg gaa ggc gcc tgg gtg gac atg acc ggt acc ctc ctg gcc tac aag 288 ala glu gly ala trp val asp met thr gly thr leu leu ala tyr lys aac tgg gag acg gag atc acg acg caa ccc gac ggc ggc aaa gcc gag 336 asn trp glu thr glu ile thr thr gln pro asp gly gly lys ala glu aac tgc gcc gcc ctg tct ggc gca gcc aac ggc aag tgg ttc gac aag 384 cga tgc cgc gat caa ttg ccc tac atc tgc cag ttt gcc att gtg 429 arg cys arg asp gln leu pro tyr ile cys gln phe ala ile val his glu ala ser glu asp cys ile ser gln gly gly thr leu gly thr pro gln ser glu leu glu asn glu ala leu phe glu tyr ala arg his ser val gly asn asp ala glu ile trp leu gly leu asn asp met ala ala glu gly ala trp val asp met thr gly thr leu leu ala tyr lys asn trp glu thr glu ile thr thr gln pro asp gly gly lys ala glu arg cys arg asp gln leu pro tyr ile cys gln phe ala ile val ggcccag ccg gcc atg gcc gag tca ccc act ccc aag gcc aag aag gct 49 gca aat gcc aag aaa gat ttg gtg agc tca aag atg ttc gag gag ctc 97 aag aac agg atg gat gtc ctg gcc cag gag gtg gcc ctg ctg aag gag 145 aag cag gcc tta cag act gtg gtc ctg aag ggc acc aag gtg aac ttg 193 aag gtc ctc ctg gcc ttc acc caa ccg aag acc ttc cat gag gcg agc 241 lys val leu leu ala phe thr gln pro lys thr phe his glu ala ser gag gac tgc atc tcg caa ggg ggc acg ctg ggc acc ccg cag tca gag 289 cta gag aac gag gcg ctg ttc gag tac gcg cgc cac agc gtg ggc aac 337 leu glu asn glu ala leu phe glu tyr ala arg his ser val gly asn gat gcg gag atc tgg ctg ggc ctc aac gac atg gcc gcg gaa ggc gcc 385 tgg gtg gac atg acc ggt acc ctc ctg gcc tac aag aac tgg gag acg 433 trp val asp met thr gly thr leu leu ala tyr lys asn trp glu thr gag atc acg acg caa ccc gac ggc ggc aaa gcc gag aac tgc gcc gcc 481 glu ile thr thr gln pro asp gly gly lys ala glu asn cys ala ala ctg tct ggc gca gcc aac ggc aag tgg ttc gac aag cga tgc cgc gat 529 leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp caa ttg ccc tac atc tgc cag ttt gcc att gtg gcg gccgc 570 leu leu ala phe thr gln pro lys thr phe his glu ala ser glu asp asn glu ala leu phe glu tyr ala arg his ser val gly asn asp ala asp met thr gly thr leu leu ala tyr lys asn trp glu thr glu ile thr thr gln pro asp gly gly lys ala glu asn cys ala ala leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp gln leu ggcccag ccg gcc atg gcc gcc tta cag act gtg gtc ctg aag ggc acc 49 aag gtg aac ttg aag gtc ctc ctg gcc ttc acc caa ccg aag acc ttc 97 cat gag gcg agc gag gac tgc atc tcg caa ggg ggc acg ctg ggc acc 145 his glu ala ser glu asp cys ile ser gln gly gly thr leu gly thr ccg cag tca gag cta gag aac gag gcg ctg ttc gag tac gcg cgc cac 193 pro gln ser glu leu glu asn glu ala leu phe glu tyr ala arg his agc gtg ggc aac gat gcg gag atc tgg ctg ggc ctc aac gac atg gcc 241 ser val gly asn asp ala glu ile trp leu gly leu asn asp met ala gcg gaa ggc gcc tgg gtg gac atg acc ggt acc ctc ctg gcc tac aag 289 ala glu gly ala trp val asp met thr gly thr leu leu ala tyr lys aac tgg gag acg gag atc acg acg caa ccc gac ggc ggc aaa gcc gag 337 asn trp glu thr glu ile thr thr gln pro asp gly gly lys ala glu aac tgc gcc gcc ctg tct ggc gca gcc aac ggc aag tgg ttc gac aag 385 cga tgc cgc gat caa ttg ccc tac atc tgc cag ttt gcc att gtg gcg 433 arg cys arg asp gln leu pro tyr ile cys gln phe ala ile val ala asn leu lys val leu leu ala phe thr gln pro lys thr phe his glu ala ser glu asp cys ile ser gln gly gly thr leu gly thr pro gln gly ala trp val asp met thr gly thr leu leu ala tyr lys asn trp glu thr glu ile thr thr gln pro asp gly gly lys ala glu asn cys ala ala leu ser gly ala ala asn gly lys trp phe asp lys arg cys arg asp gln leu pro tyr ile cys gln phe ala ile val ala ggcccag ccg gcc atg gcc gcc ctc cag acg gtc tgc ctg aag ggg acc 49 aag gtg cac atg aaa tgc ttt ctg gcc ttc acc cag acg aag acc ttc 97 cac gag gcc agc gag gac tgc atc tcg cgc ggg ggc acc ctg agc acc 145 his glu ala ser glu asp cys ile ser arg gly gly thr leu ser thr cct cag act ggc tcg gag aac gac gcc ctg tat gag tac ctg cgc cag 193 pro gln thr gly ser glu asn asp ala leu tyr glu tyr leu arg gln agc gtg ggc aac gag gcc gag atc tgg ctg ggc ctc aac gac atg gcg 241 ser val gly asn glu ala glu ile trp leu gly leu asn asp met ala gcc gag ggc acc tgg gtg gac atg acc ggt acc taagtgacga tatcctgacc 294 ala ser glu asp cys ile ser arg gly gly thr leu ser thr pro gln thr gly ser glu asn asp ala leu tyr glu tyr leu arg gln ser val ggcccag ccg gcc atg gcc aac aag ttg cat gcc ttc tcc atg ggt aaa 49 aag tct ggg aag aag ttc ttt gtg acc aac cat gaa agg atg ccc ttt 97 lys ser gly lys lys phe phe val thr asn his glu arg met pro phe tcc aaa gtc aag gcc ctg tgc tca gag ctc cga ggc act gtg gct atc 145 ser lys val lys ala leu cys ser glu leu arg gly thr val ala ile ccc aag aat gct gag gag aac aag gcc atc caa gaa gtg gct aaa acc 193 tct gcc ttc cta ggc atc acg gac gag gtg act gaa ggc caa ttc atg 241 ser ala phe leu gly ile thr asp glu val thr glu gly gln phe met tat gtg aca ggg ggg agg ctc acc tac agc aac tgg aaa aag gat gag 289 tyr val thr gly gly arg leu thr tyr ser asn trp lys lys asp glu ccc aat gac cat ggc tct ggg gaa gac tgt gtc act ata gta gac aac 337 pro asn asp his gly ser gly glu asp cys val thr ile val asp asn ggt ctg tgg aat gac atc tcc tgc caa gct tcc cac acg gct gtc tgc 385 gly leu trp asn asp ile ser cys gln ala ser his thr ala val cys gly lys lys phe phe val thr asn his glu arg met pro phe ser lys val lys ala leu cys ser glu leu arg gly thr val ala ile pro lys phe leu gly ile thr asp glu val thr glu gly gln phe met tyr val thr gly gly arg leu thr tyr ser asn trp lys lys asp glu pro asn asp his gly ser gly glu asp cys val thr ile val asp asn gly leu trp asn asp ile ser cys gln ala ser his thr ala val cys glu phe ggcccag ccg gcc atg gcc aag aaa gtt gag ctc ttc cca aat ggc caa 49 pro ala met ala lys lys val glu leu phe pro asn gly gln agt gtg ggg gag aag att ttc aag aca gca ggc ttt gta aaa cca ttt 97 acg gag gca cag ctg ctg tgc aca cag gct ggt gga cag ttg gcc tct 145 cca cgc tct gcc gct gag aat gcc gcc ttg caa cag ctg gtc gta gct 193 aag aac gag gct gct ttc ctg agc atg act gat tcc aag aca gag ggc 241 lys asn glu ala ala phe leu ser met thr asp ser lys thr glu gly aag ttc acc tac ccc aca gga gag tcc ctg gtc tat tcc aac tgg gcc 289 lys phe thr tyr pro thr gly glu ser leu val tyr ser asn trp ala cca ggg gag ccc aac gat gat ggc ggg tca gag gac tgt gtg gag atc 337 ttc acc aat ggc aag tgg aat gac agg gct tgt gga gaa aag cgt ctt 385 phe thr asn gly lys trp asn asp arg ala cys gly glu lys arg leu pro ala met ala lys lys val glu leu phe pro asn gly gln ser val thr tyr pro thr gly glu ser leu val tyr ser asn trp ala pro gly glu pro asn asp asp gly gly ser glu asp cys val glu ile phe thr asn gly lys trp asn asp arg ala cys gly glu lys arg leu val val