Patent Application: US-78579597-A

Abstract:
described is a novel in vitro method for obtaining and identifying proteins which , in their natural in vivo setting , are covalently modified after translation . to identify novel isoprenylated proteins for subsequent biochemical study , colony blots of a glycine max cdna expression library were 3 h ! farnesyl labeled in vitro . proteins identified by this screen contained several different carboxy - termini that conform to consensus farnesylation motifs . these proteins included known farnesylated proteins and several novel proteins , two of which contained 6 or more tandem repeats of a hexapeptide having the consensus sequence e or g ! g or p ! ek p or k ! k . expression library screening by direct labeling can thus be adapted to recover and identify isoprenylated proteins as well as proteins with other post - translational modifications . this identification and recovery further enables the recovery of transformants containing dna encoding the proteins , as well as the raising of antibodies to the proteins .

Description:
for the purpose of promoting an understanding of the principles of the invention , reference will now be made to certain preferred embodiments thereof and specific language will be used to describe the same . it will nevertheless be understood that no limitation of the scope of the invention is thereby intended , such alterations , further modifications and applications of the principles of the invention as described herein being contemplated as would normally occur to one skilled in the art to which the invention relates . the following abbreviations are used herein : fpp , farnesyl pyrophosphate ; ftase , farnesyl : protein transferase ; ggtase , geranylgeranyl : protein transferase the ras proteins used in this study were encoded by pras1 plasmids containing a truncated yeast ras1 gene ( 1 , 27 , 28 ). in pras1 , the yeast ras1 gene is fused in the correct translational reading frame to the first 6 codons of lacz , 5 polylinker codons , 9 codons from p21v - ras sequences upstream of and adjacent to the normal p21v - ras initiation codon and the first four codons of p21v - ras ( 27 , 28 ). the 3 &# 39 ; end of the yeast ras1 gene was subsequently truncated and altered by site - directed mutagenesis to encode the carboxy - terminal peptide caim , cail , or svls ( 1 ). ( permission to use these plasmids was obtained from merck , sharpe and dohme research laboratories , west point , pa .) yeast ras1 was highly expressed as soluble protein ( approx 10 - 20 % of total protein ) in e . coli strain rr1 laci q ( 1 , 28 ). yeast ras proteins with different carboxy - termini were expressed at similar levels . expressed proteins were purified from e . coli cell extracts to greater than 90 % purity by medium pressure liquid chromatography on a deae - sepharose column ( pharmacia ), and were eluted at approx 0 . 1 m in a 0 - 1 . 0 m nacl gradient . purified proteins were dot - blotted on nytran plus ( schleicher and schuell ) membrane filters at different concentrations in tris buffered saline ( tbs , 50 mm tris , ph 8 . 0 , 150 mm nacl ) containing 1 mg / ml bovine serum albumin ( bsa ), and farnesyl labeled as described below . between 1 × 10 3 and 3 × 10 4 e . coli strain rr1 laci q transformants ( electroporation method , bio - rad gene pulser protocol ) were spread on a 82 mm diameter nitrocellulose filter , which was overlaid on an lb plate containing 150 mg / l ampicillin ( amp ) and incubated overnight at 37 ° c . a single replica was made onto a dry nytran plus filter ( 29 ), and fusion protein synthesis was induced by placing the replica on an lb plate containing 150 mg / l amp and 1 mm isopropyl - b - d - thiogalactopyranoside ( iptg ) for 6 hours at 37 ° c . original filters were then incubated for 6 hours on fresh lb plates containing 150 mg / l amp , placed on whatman # 1 filter papers soaked in 10 % glycerol , stored at - 80 ° c ., and later used to recover positive clones . protein blots of lysed bacterial colonies were made by incubating replica filters sequentially on blotting paper soaked in the following solutions : a ) 150 mm nacl , 100 mm tris , ph 8 . 0 , 5 mm mgci 2 , 2 mg / ml dnase i , 50 mg / ml lysozyme , 20 min b ) 150 mm nacl , 100 mm naoh , 0 . 1 % sds , 5 min c ) 150 mm nacl , 100 mm tris hcl , ph 6 . 5 , 5 min . filters were then air dried and stored up to 1 week at 25 ° c . before blocking and prenyl labeling . air dried filters were blocked by gently shaking in tbs containing 20 mg / ml bsa and 0 . 05 % tween 20 for 1 - 2 hours at 25 ° c . the blocking solution was changed 2 - 3 times . the filters were then rinsed , the bound proteins labeled with 3 h ! farnesol by incubation in the presence of 3 h ! fpp and the ftase present in a tobacco protein extract , and unincorporated label was washed away . the rinsing , labeling , and washing were done by vigorous rotary shaking at 30 ° c . in the following series of solutions : 1 ) tbs , 10 ml per 82 mm dia filter , 4 × 5 min ; ( 2 ) prenylation buffer : 50 mm hepes ph 7 . 5 , 20 mm mgcl 2 , 5 mm dithiothreitol , 5 ml / filter , 5 min ; ( 3 ) protein extract of a 2 - day - old tobacco suspension culture ( ref . 20 ) with prenylation buffer added fresh from a 4 . 4 × concentrate , 1 ml / filter , 5 min ; ( 4 ) labeling solution as in # 3 containing 4 mci / ml 3 h ! fpp ( 15 ci / mmol , american radiochemical ), 1 ml / filter , 1 hour ; ( 5 ) 1 ml / filter solution # 3 added to labeling solution , 1 hour ; ( 6 ) tbs , 10 ml / filter , 6 × 5 min ; ( 7 ) 50 % ethanol , 10 ml / filter , 3 × 5 min ; ( 8 ) 100 % ethanol , 10 ml / filter , 3 × 5 min . filters were then air dried , dipped in amplify ( amersham ) fluorographic reagent , placed on plastic film backing , dried 1 hour at 50 ° c ., and fluorographed 7 - 30 days at - 80 ° c . with pre - flashed kodak xar5 film . the vector ( phep1 ) was constructed by inserting the 141 bp hind iii / pvu ii fragment of puc 19 into hind iii / nde i - cut pras1 , generating multiple cloning sites downstream of the orf to which the yeast ras1gene had been fused ( 27 ). soybean cell culture cdna ( 30 ), amplified once in pbluescript sk ii + ( stratagene ), was excised with hind iii ( 5 &# 39 ;) and sac i ( 3 &# 39 ;) and ligated into the plasmid phep1 . cultures were grown 12 - 15 hours at 37 ° c . in lb containing 150 mg / l ampicillin and 1 mm iptg and proteins were extracted ( 31 ). 2 . 5 ml of e . coli protein extract was farnesyl - labeled in a 12 . 5 ml volume by proteins extracted from either soybean or tobacco cell suspension cultures ( 20 ). sds polyacrylamide gels ( 14 %) of electrophoretically separated , labeled proteins were soaked in amplify ( amersham ), dried and fluorographed with kodak xar5 film 7 - 30 days at - 80 ° c . the molecular mass of the fusion protein does not necessarily correspond to that of the corresponding plant protein , since the cdna coding region may not all be present . furthermore , vector - encoded amino acids comprise 3 kda of the fusion protein molecular mass . dideoxy nucleotide sequencing of both strands of cdnas was according to sequenase ( u . s . biochemical ) protocols . oligomers were synthesized to prime cdna sequences distal to vector priming sites . database searches were conducted with the fasta and tfasta programs ( 32 ). as a first step toward identifying soybean isoprenylated proteins , the sensitivity and specificity of in vitro prenylation of blotted proteins were tested . plasmid - encoded yeast ras1 proteins with the carboxy - terminal sequences caim , cail , or svls were prepared from e . coli clones obtained from mark marshall ( 1 ). yeast and tobacco prenyltransferases show similar specificity toward these carboxy - termini : ras - caim is preferentially farnesylated , ras - cail is readily geranylgeranylated but not farnesylated , and ras - svls is not a substrate for prenylation ( 1 , 20 ). as shown in fig1 a , the lowest protein concentration detected by radiolabeling protein blots with 3 h ! fpp in the presence of a tobacco extract was 0 . 12 mmol m - 2 for purified ras - caim and ≦ 12 mmol m - 2 for ras - cail . lacz fusion proteins expressed in e . coli bacteriophage 1 vectors are synthesized at up to 100 pg / plaque ( 0 . 001 mmol m - 2 for a protein of 100 , 000 da ) ( 29 ). this is about two orders of magnitude less than the minimum ftase substrate concentration detectable in our assays ( fig1 a ). since expression levels are generally higher in plasmid libraries , we tested the feasibility of detecting prenylated proteins in such a library using colony blots of the plasmid - encoded ras proteins described above . as shown in fig1 b , blotted proteins of e . coli colonies expressing ras - caim were labeled by in vitro prenylation , demonstrating that bacterially - expressed ftase substrates could be detected by this method . colony blots of bacteria expressing proteins which are not ftase substrates ( ras - cail and ras - svls ) had very weak or undetectable signals after prolonged autoradiography . these signals were readily distinguishable from the ras - caim positive control ( fig1 b ). thus , labeling of e . coli protein blots in the presence of 3 h ! fpp was specific for colonies expressing a ftase substrate , indicating that the protein blotting and prenylation methods might be used to specifically identify farnesylated proteins among those encoded by a cdna library . since high levels of soluble recombinant protein were obtained from the vector expressing yeast ras ( 1 , 27 , 28 ), we constructed a cdna expression library in this vector by inserting soybean cdnas into the restriction site ras1 had occupied . approximately 3 × 10 5 cdna - containing clones were screened by prenylating replica colony protein blots ( 29 ) with 3 h ! fpp in the presence of a tobacco extract , resulting in the identification of 158 putative positive clones ( see fig2 a ). cells on areas of filters where positive signals were obtained were re - screened . of the colonies that produced a signal in the first screen , 72 % were positive in the second screen . molecular masses of prenylated proteins from positive colonies were determined ( fig2 b ) by prenylating e . coli culture extracts with 3 h ! fpp in the presence of a tobacco or soybean cell culture extract , and separating the proteins by sds - page . one soybean cdna ( glycine max farnesylated protein 1 , or gmfp1 , fig3 a ) encoded an 86 amino acid prenylated protein highly homologous ( 63 % amino acid identity ) to the carboxy - terminus of the atriplex stress - induced protein anj1 , the only plant protein known to be prenylated in vivo ( 21 , 22 ). a similar clone ( not shown ) with approximately 95 % peptide identity to gmfp1 and designated gmfp9 , encoded an approximately 300 amino acid peptide homologous to anj1 . anj1 is structurally related to e . coli dnaj , and is thought to function as a molecular chaperone ( 22 ). two cdnas encoding similar proteins , gmfp2 and gmfp3 , ( fig3 b ) predominated among the cdnas encoding farnesylated proteins . sequence analyses and dna hybridizations ( 29 ) of positive clones revealed that more than 90 % of the cdna clones selected by protein farnesyl - labeling were structurally similar to gmfp2 and gmfp3 . dna and deduced peptide sequences of gmfp2 and gmfp3 were not similar to other sequences in the genbank database . both deduced peptide sequences had basic regions containing 6 or 8 repeats of a hexapeptide having the consensus sequence e or g ! g or p ! ek p or k ! k . most of these repeats were separated by 1 or 2 amino acids ( fig3 b ). cysteine residues 110 and 115 of gmfp2 and 134 of gmfp3 are possible sites for palmitoylation . gmfp6 ( fig3 c ) has structural features similar to gmfp2 and gmfp3 : the c - terminal peptide eenpxgcxix is conserved ( seq id no : 4 - 6 ), as well as a repeat motif rich in glutamate , lysine and proline . several different farnesylated proteins identified from arabidopsis and tobacco expression libraries have these same two structural features suggesting that they are functionally related . sequences at the 5 &# 39 ; and 3 &# 39 ; ends ( 150 - 350 bases ) of four additional farnesylated protein - encoding cdnas did not show any similarity to cdnas described here , or to each other 3 . three of these prenyl - labeled proteins are shown in fig2 b ( gmfp4 , - 5 , and - 7 ). thus , other types of soybean prenylated proteins , whose characterization is in progress , were detected by this procedure . the carboxy - terminal peptides found on prenylated proteins identified by this screen conform to the consensus caax motif recognized by ftases . furthermore , these peptides are identical or very similar to carboxy - terminal peptides known to be farnesylated in vivo . the carboxy - terminus of gmfp3 ( ciim ) is identical to the farnesylated carboxy - terminus of human k - ras ( c - 4a ) ( 33 , 34 ). likewise , the carboxy - terminal tetrapeptide of gmfp1 , caqq is identical to that of anj1 ( 21 ). the carboxy - terminal tetrapeptide of gmfp2 ( ctim ) is similar to that of chicken prelamin a ( csim ) ( 16 ). gmfp6 had the carboxy - terminal caax sequence cvic , identical to that of s . pombe ras ( 35 ). the carboxy - terminus cvvm ( identical to that of n - ras , 36 ) was encoded 170 bases upstream of the poly ( da ) tail of gmfp7 ( fig2 b ). our results indicate that this screening method will facilitate the identification of novel prenylated proteins in other systems . ggtase substrates may also be identified by this method . since the method does not require sensitive detection of proteins (≧ 0 . 12 mmol m - 2 ), it is expected that it can be adapted to identify plasmid - encoded proteins with properties other than the covalent linkage of an isoprenoid . properties conferred by a small linear domain , such as a protein kinase phosphorylation site ( 37 ), are expected to be detectable by this method . in addition , other protein characteristics that are retained after protein blotting , such as autophosphorylation ( 38 - 40 ), calcium binding ( 41 ), and binding by pharmaceuticals , herbicides or insecticides , may also be detectable on colony blots in accordance with the invention . because the hydrophobic prenyl moiety generally promotes membrane association , it is reasonable to expect that many of these farnesylated soybean proteins are membrane localized . the basic repeats found in the deduced gmfp2 and gmfp3 peptides suggest that they could be located in the nucleus and may bind nucleic acids . our results show that plants contain a diverse array of genes encoding farnesylated proteins , and suggest that fundamental differences in the identities of farnesylated proteins may exist between plants and other eukaryotes . the following references are hereby incorporated by reference as if each had been individually incorporated by reference and fully set forth . 1 . moores , s . l , schaber , m . d ., mosser , s . d ., rands , e ., o &# 39 ; hara , m . b ., garsky , v . m ., marshall , m . s ., pompliano , d . l . and gibbs , j . b . 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( 1986 ) cell 47 , __________________________________________________________________________ # sequence listing - ( 1 ) general information :- ( iii ) number of sequences : 5 - ( 2 ) information for seq id no : 1 :- ( i ) sequence characteristics :# amino acids ) length : 86 # amino acidb ) type : ( d ) topology : linear - ( ii ) molecule type : protein - ( xi ) sequence description : seq i - # d no : 1 :- leu tyr ile gln phe asn val asp phe pro as - # p ser gly phe leu # 15 - ser pro asp gln cys gln leu leu glu lys va - # l leu pro gln lys # 30 - ser ser lys his val ser asp met glu leu as - # p asp cys glu glu # 45 - thr thr leu his asp val asn phe lys glu gl - # u met arg arg lys # 60 - gln gln gln gln tyr arg glu ala tyr asp gl - # u asp asp asp glu # 75 - pro ser gly gln arg val gln cys ala gln gl - # n # 85 - ( 2 ) information for seq id no : 2 :- ( i ) sequence characteristics : ( a ) length : 84 - # amino acids # amino acid ) type : ( d ) topology : linear - ( ii ) molecule type : protein - ( xi ) sequence description : seq id no : - # 2 :- met tyr ile his phe thr val glu phe pro as - # p ser leu asn pro # 15 - asp gln val lys ser leu glu ala ile leu pr - # o pro lys pro ser # 30 - met ser leu thr tyr met glu leu asp glu cy - # s glu glu thr thr # 45 - leu his asn val asn ile glu glu glu met ly - # s arg lys gln thr # 60 - gln ala gln gln glu ala tyr asp glu asp as - # p glu pro ala gly # 75 - gly gln arg val gln cys ala gln gln 80 - ( 2 ) information for seq id no : 3 :- ( i ) sequence characteristics :# amino acids ) length : 130 # amino acidb ) type : ( d ) topology : linear - ( ii ) molecule type : protein - ( xi ) sequence description : seq id no : 3 :- lys ala glu lys pro lys thr glu pro glu ly - # s lys lys asp gly # 15 - gly gly glu lys pro lys glu glu pro glu ly - # s lys lys asp gly # 30 - gly glu lys pro lys pro gly pro glu lys pr - # o lys asp lys pro # 45 - thr pro ala pro leu pro val gln pro his il - # e ala ala pro met # 60 - ala val pro val gly met leu tyr ala pro pr - # o pro cys tyr gly # 75 - gly arg pro val gly pro gly tyr glu tyr gl - # y gly pro met leu # 90 - cys tyr asp gly tyr tyr ala arg pro val ty - # r asp ser tyr ser # 105 - gly gly arg pro cys tyr gly asn arg cys as - # p gln tyr phe ser # 120 - glu glu asn pro gln gly cys thr ile met # 130 - ( 2 ) information for seq id no : 4 :- ( i ) sequence characteristics :# amino acids ) length : 154 # amino acidb ) type : ( d ) topology : linear - ( ii ) molecule type : protein - ( xi ) sequence description : seq i - # d no : 4 :- lys pro lys ala glu pro glu lys lys lys as - # p gly gly gly glu # 15 - lys pro lys ala glu pro glu lys lys lys as - # p gly gly gly glu # 30 - lys pro lys ala glu pro glu lys lys lys as - # p gly gly glu lys # 45 - pro lys gly asp ala pro lys lys glu ala gl - # u lys pro lys pro # 60 - gly pro glu lys pro lys asp lys pro ala pr - # o ala pro leu pro # 75 - val gln pro his met ala ala pro met ala va - # l pro val gly met # 90 - leu tyr ala pro pro pro cys tyr glu gly ar - # g pro val gly pro # 105 - gly tyr glu tyr gly gly pro met phe cys ty - # r asp gly tyr tyr # 120 - ala arg pro val tyr asp ser tyr gly gly gl - # y arg pro cys tyr # 135 - val asn arg gly asp gln tyr phe ser glu gl - # u asn pro gln gly # 150 - cys ile ile met - ( 2 ) information for seq id no : 5 :- ( i ) sequence characteristics :# amino acids ) length : 74 # amino acidb ) type : ( d ) topology : linear - ( ii ) molecule type : protein - ( xi ) sequence description : seq i - # d no : 5 :- lys leu arg lys phe cys his val glu ile le - # u ser val gly pro # 15 - ala lys glu glu pro lys lys glu glu lys ly - # s pro glu ala lys # 30 - lys asp pro lys glu glu tyr ala glu leu le - # u lys val val glu # 45 - ala asn tyr tyr gln thr arg his leu gln ty - # r pro tyr tyr tyr # 60 - ser arg thr val glu glu asn pro thr gly cy - # s val ile cys # 70__________________________________________________________________________