WO2007076974A2 - Procede de production d'une proteine chimiquement modifiee - Google Patents

Procede de production d'une proteine chimiquement modifiee Download PDF

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WO2007076974A2
WO2007076974A2 PCT/EP2006/012434 EP2006012434W WO2007076974A2 WO 2007076974 A2 WO2007076974 A2 WO 2007076974A2 EP 2006012434 W EP2006012434 W EP 2006012434W WO 2007076974 A2 WO2007076974 A2 WO 2007076974A2
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protein
modified
intein
group
modification
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PCT/EP2006/012434
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German (de)
English (en)
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WO2007076974A3 (fr
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Henning Mootz
Thomas Kurpiers
Christina Ludwig
Steffen Brenzel
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Philipps-Universität Marburg
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Publication of WO2007076974A3 publication Critical patent/WO2007076974A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a two-stage process for the preparation of a chemically modified protein.
  • NCL Native chemical ligation
  • EPL expressed protein ligation
  • the modification of cysteines is not regioselective, ie, if more cysteines are present in the protein, as is almost always the case with larger proteins, all cysteines will undergo this reaction , This is often problematic, for example when another cysteine is important for the activity of the protein or when a regioselective and stoichiometrically defined modification is to be made. Therefore, a method is presented according to the invention, in which the synthesis of the modified protein is divided into two process steps. Only in the first step, the modification of the intein auxiliary protein, the modification of the cysteine plays a role. Since this step is a well-defined intein auxiliary protein that contains only a single cysteine, this eliminates the problem of lack of selectivity.
  • the subject of the process according to the invention is the preparation of a chemically modified protein in which in a first method step, an auxiliary protein carrying a reactive functional group consisting of an intein fragment and an exine sequence, modified with a group to be introduced into the target protein, and
  • a target protein which is to be modified and fused with a complementary intein fragment is reacted so that the modified extein sequence is linked to the target protein.
  • an auxiliary protein which contains an integer C is used in the method according to the invention in the first method step.
  • the method according to the invention can also be designed such that in the first method step a protein is used which contains an intein N and the target protein used in the second method step is fused with an intein C.
  • the reactive functional group of the auxiliary protein is an SH group. Since the SH group reacts with a maleimide or a haloacetamide in a very simple and clear manner, it is very advantageous to introduce the group to be introduced into the target protein bound to a maleimide or a haloacetamide having one SH group carrying auxiliary protein to modify.
  • the group to be introduced into the target protein if it is to serve as a label, e.g. a fluorophore residue, a biotin residue, an oligonucleotide residue or a radioactive residue.
  • auxiliary protein If an integer C is used as auxiliary protein, then the extra-sequence must consist of at least two amino acids. If, on the other hand, an auxiliary protein is used which contains an intein N, then the extein sequence contained in the auxiliary protein must have at least one amino acid.
  • the crucial step of the present invention is the use of the conventional chemical modification of cysteine in proteins, without the To accept the disadvantages of non-specificity.
  • the reaction is illustrated by the reaction scheme shown in Fig. 1:
  • a protein (auxiliary protein, "Intein C-cystag”) containing only one cysteine is modified by the method described above, and the resulting protein modification, the protein "In C-cystag” modification, is then transformed into purified form reacted with the actual target protein, which in turn was previously partially fused with an intein ("target protein Intein N").
  • target protein Intein N an intein
  • the intein N and intein C halves then interact to form the active intein, which cuts itself out autocatalytically through the process of protein splicing, thereby involving the fused sequences ("target protein” and “cystag modification”) a peptide bond linked together.
  • the principle of the invention is illustrated in FIG.
  • the advantage of this reaction is that additional cysteines contained in the "target protein” are unaffected by this reaction, so the chemical reaction at the "cystag” is regioselectively linked to the target protein, at the C-terminus of the target protein.
  • the "cystag” is a sequence of a few amino acids, which has the advantage that the further modification of the target protein with only a few amino acids, which is worth to carry out the method, is minimal
  • the C-extein sequence may also have a longer sequence, such as, for example, an affinity chromatography a larger part of a protein.
  • intein parts N and C are known and have been developed starting from the Ssp DnaB Intein.
  • two different intein N and intein C parts of the Ssp DnaB mini-link were obtained by genetic engineering methods corresponding to a split of the intein at two different sites.
  • cleavage sites are the sites SO and S1 as reported by Sun et al. in the Journal of Biological Chemistry, (2004), 279: 35281 to 35286.
  • the cleavage site SO was first described by Wu et al., Journal of Biological Chemistry, (1998), 1387: 422-432.
  • the intein halves intein C (SO) and intein C (S1) were prepared as purified, recombinant polypeptides with the "cystag" as C-extein sequence In the auxiliary protein Intein C (SiO), the remaining cysteine at position 50 of the intein
  • the complementary intein halves intein N (SO) and intein N (S1) were fused to the respective target protein.
  • the present invention is not limited to the aforementioned inteins but may be extended to other inteins. All inteins which have no cysteine as the catalytic N- or C-nucleophile but have a serine or threonine and which can be cleaved into active intein N and intein C halides are suitable for this purpose. Further cysteines occurring within the intein must then be removed by mutagenesis if necessary.
  • the method according to the invention can also be carried out by linking an intein N-half to a cystag (see FIG. 1b). Then the target protein can be modified at its N-terminus.
  • a cleaved intein whose catalytic N-nucleophile is serine, and which otherwise contains no cysteine residues, is the Psp-GBD PoM intein described by Southworth et al., EMBO Journal (1998), 17: 918-926 This integer half complies with all requirements if it is appropriately provided with a cystag and modified according to the invention.
  • the products produced by the process of the invention can be distinguished from the products made by the chemical ligation method.
  • the modification reagent for example, fluorescein-maleimide
  • the modification reagent is at the side chain a cysteine residue at the C-terminus or N-terminus of the protein coupled, while possibly. Additional cysteine present in the protein unmodified. This is usually not the case with the previously known methods.
  • the hitherto customary biophysical probes such as fluorophores, biotin, oligonucleotides can be linked to proteins or proteins can be attached to solid phases such as chips and microarrays as well as to synthetic cofactors, but also therapeutically and diagnostically interesting proteins can be produced.
  • These usually have to carry the numerous post-translational modifications that the human body makes to them in order to be biologically and medically effective.
  • synthetic modification of proteins which contribute to the stability and thus longer residence time in the body. Such modifications can also be introduced according to the invention into proteins using the present method.
  • Glycosylations Glycosylations, phosphorylations, acetylations, isoprenylations, farnesylations, lipoylations, myristoylations, palmitoylations, polyethylene glycolylations and the incorporation of chelating agents for metal ions such as technetium for the visualization or irradiation of tumors.
  • pMST Ssp DnaB mini- Intein from pMST (Wu et al., Biochimica et Biophysica Acta 1387 (1998) 422-432).
  • pMST served as a template for a polymerase chain reaction (PCR), wherein the C-terminal part of the DnaB intein (DnaB c ) of the cleavage site SO correspondingly using the oligonucleotides 5'-ATACCATGGGCACTAGTTCACCAGAAATAGAAAAGTTGTC-3 '(recognition sequences of the restriction endonucleases ⁇ / col and Spei are underlined) and ⁇ '-ATAGGTACCAGATCTAATACTGTTATGGACAAT-GATGTC-S '(Kpn ⁇ , BgIW) was amplified.
  • PCR polymerase chain reaction
  • the purified PCR fragment was then cut with ⁇ / col and BgIW.
  • the resulting fragment was subsequently ligated by means of T4 DNA ligase into the similarly prepared vector pQE60 (Qia gene).
  • the vector pTK048 was obtained.
  • the starting plasmid pTK048 served as a DNA template for a PCR with the two oligonucleotides oHM116 (5'-ATA CCA TGG GCA CTA GTT CAC CAG / VAA TAG AAA AGT TGT C-3 ', ⁇ / col, Spei) and oTK14 (5 '-ATA AGA TCT ACC GCA ACC CTG TTC GAT ACT GTT ATG GAC AAT GAT G-3', BgIW) containing the DnaB c coding fragment with the extein SEQUGCGRSHHHHH sequence.
  • Plasmids pTK048 and pTK049 were cut with the restriction enzymes Spei and Hin ⁇ W ⁇ and the insert ligated into the Xba ⁇ and HindWl-treated vector pHM45 (Mootz et al., J. Am. Chem. Soc.
  • the expression plasmids for the fusion proteins with the N-terminal intein fragment DnaB N were prepared from plasmid pSB13.
  • pTK056 could be obtained (coding for MBP-DnaB N -His 6 ).
  • pTK060 (encoding TyrA-PCP-DnaB N -His 6 ) was recovered after removal of the MBP-encoding fragment in pTK056 by ⁇ / col and / ⁇ coRI treatment followed by ligation with a fragment encoding TyrA-PCP.
  • the latter was amplified by PCR with the oligonucleotides oTL02 (5'-TTT TCC ATG GCG TTG TCC GAG ATC ACC ATG-3 ', ⁇ / col) and oTK19 (5'-ATA GAA TTC TCC GCT CGT GGC GAC ATA CTG GGC CAA CGC- 3 ', EcoRI) from the construct pCLA-PCP2b1 amplified.
  • the construct pCLA-PCP2b1 was isolated from Bacillus brevis chromosomal DNA using primers OTL02 (5'-TTT TCC ATG GCG TTG TCC GAG ATC ACC ATG-3 ', ⁇ / col) and OTL03 (5'-AAA AAG ATC TCG TGG CGA CAT ACT GG-3 ', BgIW) in the vector pQE60 cloned.
  • TyrA-PCP is the tyrosine activating adenylation domain and corresponding non-ribosomal peptidyl carrier protein domain (PCP).
  • Tyrocidin synthetase TycC3 (Mootz and Marahiel, J. Bacteriol. (1997) 179: 6843-6850). This protein contains six cysteine residues, which would also lead to unwanted side reactions under conventional sulfhydryl labeling of proteins.
  • E. coli BL21 cells were transformed with the corresponding plasmids pTK053, pTK054, pTK055, pTK056 and pTK060.
  • the resulting expression strains were grown in a preculture (LB medium with ampicillin (100 ⁇ g / ml) and for the MBP-containing proteins with additional 0.2% glucose) overnight at 37 ° C. and 250 rpm. 6 ml of the respective preculture were used to inoculate 600 ml of the same medium. After the cell cultures at 37 0 C and 250 rpm to an optical density (OD 6 oo nm) reached approximately 0.7, the temperature was lowered to 30 ° C and the production of proteins by the addition of isopropyl - /? - D -thiogalactopyranoside in a final concentration of 0.4 mM.
  • the cells were pelleted by centrifugation after 3-4 hours, the supernatant discarded, and the cell pellet resuspended in wash buffer (50 mM Tris, 300 mM NaCl, pH 8.0) with 5 mM imidazole.
  • wash buffer 50 mM Tris, 300 mM NaCl, pH 8.0
  • the digestion of the cells was carried out by 2 passes with an emulsifier (A vestin EmulsiFlex C-5).
  • the insoluble cell constituents were separated by centrifugation at 30,000 g (15 min) and the soluble fraction was then applied to an equilibrated Ni 2+ -NTA gravity flow column (Qiagen company, bed volume about 2 ml).
  • the washing steps were carried out with 50 ml washing buffer with 5 mM imidazole, 30 ml washing buffer with 20 mM imidazole and 10 ml washing buffer with 40 mM imidazole.
  • the bound proteins were then eluted with wash buffer with 250 mM imidazole.
  • the fusion proteins which also contained MBP, were then dialyzed against amylose buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.4) and applied to an equilibrated amylose column (New England Biolabs, bed volume about 4 mL).
  • the proteins were finally eluted with amylose buffer with 10 mM maltose.
  • the pooled fractions with the respective purified protein were assayed against splice buffer (50 mM Tris, 300 mM NaCl, 2 mM DTT, 1 mM EDTA, 10% glycerol, pH 7.0) or against modification buffer (20-50 mM phosphate, 150 mM NaCl , 7.2), dialyzed 10% glycerol, pH, and then frozen until further use at -80 0 C.
  • splice buffer 50 mM Tris, 300 mM NaCl, 2 mM DTT, 1 mM EDTA, 10% glycerol, pH 7.0
  • modification buffer 20-50 mM phosphate, 150 mM NaCl , 7.2
  • the splicing reactions were carried out at 25 0 C in Spl bosspuffer wherein DTT was added fresh immediately before the reaction (final concentration 2 mM).
  • An intein construct was placed in splice buffer and the reaction started by addition of the corresponding other infusion protein. The final concentration of proteins was adjusted to 2 ⁇ M unless otherwise stated.
  • the splicing reactions were stopped by addition of 10 ⁇ l of 4x SDS buffer (500 mM Tris / HCl, pH 6.8, 8% SDS, 40% glycerol, 20% ⁇ -mercaptoethanol, 5 mg / mL bromophenol blue) to 30 ⁇ L aliquots of the splice mixture ,
  • the modification reactions were carried out with the corresponding proteins and the respective modification reagent at 25 ° C. and protected from light.
  • the protein to be modified was used in concentrations of 10-40 ⁇ M, mixed with the 10-fold molar excess of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) in modification buffer and then mixed with a 10-25-fold molar excess of modification reagent.
  • Modification reactions were performed after 120 min by addition of 1 mM (final concentration) DTT, 1 mM (final concentration) of ⁇ - Mercaptoethanol, or the 0.25-fold volume 4xSDS sample buffer stopped.
  • the modification reaction with various reagents are described in detail below.
  • the modification reaction was stopped with 1 mM DTT (10 min) and used with the corresponding N-terminal intein construct in splice buffer for splicing reaction.
  • the modification reactions were stopped with the 0.25-fold volume of 4x SDS sample buffer, applied directly to an SDS gel, and then the gel was applied on a UV screen (312 nm) or using Coomassie -Brilliant-Blue staining analyzed. Fig.
  • a fresh 10 mM stock solution of BM in modification buffer was prepared.
  • the proteins TK095 and TK097 to be modified were each initially introduced in concentrations of 10-40 ⁇ M and added with 10 times the molar amount of TCEP for 5 minutes 25 ° C preincubated. Subsequently, the 15-fold molar amount of BM was added and the reaction mixture incubated for a further 2 hours at 25 ° C protected from light.
  • Fig. 4 schematically shows the modification reaction of the Cys-tag constructs with BM.
  • the reaction mixture was dialyzed against avidin column buffer (50 mM Tris, 150 mM NaCl, pH 7.5) to separate excess BM.
  • the dialyzed protein was loaded onto an equilibrated soft-link soft-elute avidin column (Promega), washed with 10-fold column volumes of avidin column buffer and the biotinylated protein then eluted with avidin column buffer (+ 10 mM biotin).
  • the SDS-gel of purification, as well as the mass determination of the eluted, biotinylated protein TK095 are shown in Fig. 5.
  • the modification reagent was dissolved in DMF and then adjusted to 10 mM with modification buffer (final concentration DMF 2% (v / v) in modification buffer).
  • the proteins to be modified were reduced with 10 times the molar amount of TCEP for 5 minutes, then mixed with 25 times the molar amount of 5-IAF and incubated protected from light for a further 2 hours.
  • the analysis of the course of the reaction was carried out as described for the modification with FM and is shown in FIG.
  • the modified protein TK095 was then used for the splicing reaction.
  • splicing reaction For the splicing reaction, the unmodified protein TK095 was presented in splicing buffer. The reaction was then started by adding the protein TK115, or TK117. The final concentrations of the two proteins were 2 ⁇ M each. After different time points, an aliquot was removed from the reaction and the reaction was stopped by adding 4xSDS sample buffer. stops.
  • the splicing reaction of TK095 and TK115 is shown schematically in Fig. 8 and the associated analysis by SDS-PAGE followed by densitometric evaluation in Fig. 9.
  • the C-terminal intein construct TK095 was modified with 5IAF, FM, or BM as described above and optionally purified.
  • the modified protein was then introduced into splicing buffer and the splicing reaction was started by addition of the N-terminal intein construct (TK115 or TK117).
  • the concentration of the two proteins was 2 or 4 ⁇ M in each case.
  • samples were taken for analysis by SDS-PAGE.
  • the splicing reaction was monitored by monitoring the bands on a UV screen (for the 5IAF and FM modified proteins only), by Coomassie staining, and by mass spectrometric analysis.
  • Fig. 10 shows schematically the reaction of modified TK095 with TK115.
  • Figures 11a and 11b show the analysis of the reactions by UV irradiation of the reaction mixture separated by SDS-PAGE.
  • the modified target protein could be detected by the corresponding band in the SDS-PAGE gel, as shown in Fig. 11 by the splicing reactions of TK095 modified with FM (Fig. 11a) and 5IAF (Fig. 11 b).
  • Figure 11c shows a mass spectrometric analysis (ESI-MS) of the reaction mixture of TK115 with TK095 / FM.
  • ESI-MS mass spectrometric analysis
  • the fluorescein-modified splicing product MBP-Cys (FM) -His is clearly detected.
  • Figure 12 shows the analysis of the response of TK117 with TK095 / 5IAF.
  • TyrA-PCP-Cys (5IAF) -His carries the fluorescent marker.
  • Figure 13 shows the analysis of the splicing reactions using Coomassie stained SDS-PAGE gels of biotinylated and purified proteins according to Figure 5 TK095 / BM and TK097 / BM with TK115 and TK117.
  • FIGS. 2 to 13 are shown in FIGS. 2 to 13:
  • Figure 2 shows the schematic representation of the modification reaction of the C-terminal intein Cys-tag constructs TK095 and TK097 with fluorescein-5-maleimide (FM).
  • FIG. 3a shows SDS-PAGE
  • Figure 4 shows the schematic representation of the modification reaction of the C-terminal intein Cys-tag constructs TK095 and TK097 with maleimide PEO 2 - biotin (BM).
  • Figure 5 a shows Coomassie stained SDS gel of purification
  • Figure 5b shows mass determination of the eluted, biotinylated protein TK095 / BM.
  • ESI-MS Mass spectrometric analysis
  • FIG 6 shows the schematic of the modification reaction with 5-iodoacetamidofluorescein (5IAF).
  • FIG. 7a shows SDS-PAGE
  • Figure 8 shows the schematic of the splicing reaction of TK115 and unmodified TK095.
  • the two resulting N- and C-terminal intein splice products are also shown.
  • Figure 9 b shows densitometric analysis of the splice bands, whereby the decrease in the educt band of TK115 at 56.4 kDa and the increase in the E5 edge of the splicing product MBP-Cys-His were observed at 44.8 kDa.
  • Figure 10 shows the schematic representation of the splicing reaction of TK115 with TK095 previously modified with 5IAF.
  • Figure 11 a) shows SDS-PAGE of the splicing reaction of TK115 with TK095 previously modified with fluorescein-5-maleimide, showing the modified protein TK095 / FM and the splicing reaction with TK115 after 0, 1 and 2h.
  • FIG 11 b shows SDS-GeI of the splicing reaction of TK115 with TK095 modified with 5-iodoacetamidofluorescein.
  • the SDS gels were analyzed on a UV screen.
  • the newly arising splicing product MBP-Cys (FM, or 5IAF) -HiS is highlighted by an arrow.
  • Figure 12 shows SDS-PAGE analysis of the splicing reaction of TK117 with the modified protein TK095 / 5IAF. In each case, the progress of the splicing reaction after one hour is shown. Both the gel before Coomassie staining under UV light (left) and the same Coomassie stained SDS gel (right) are shown.
  • Figure 13 shows SDS-PAGE analysis (Coomassie staining) of the splicing reaction of the purified, biotinylated proteins TK095 and TK097. Shown are the splicing reactions after 2 h. The splicing reactions with TK115 (left) show the respective newly formed bands of the splicing products MBP-SIEQGC (BM) GRS-HiS (45.3 kDa) and MBP-SIRSC (BM) G-HiS (44.9 kDa) , In the case of splicing reactions with TK117 (right), the splice products of the calculated quantities (TyrA-PCP-SIEQGC (BM) GRS-His (69.2 kDa) or TyrA-PCP-SIRSC (BM) G-His ( 68.9 kDa)).

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Abstract

Procédé de production d'une protéine chimiquement modifiée, qui consiste, lors d'une première étape, à modifier une protéine auxiliaire réactive portant des groupes fonctionnels, constituée d'un fragment d'intéine et d'une séquence d'extéine, avec un groupe à introduire dans la protéine cible, et lors d'une seconde étape, à mettre en réaction une protéine cible à modifier fusionnée avec un fragment complémentaire d'intéine de manière que la séquence d'extéine modifiée se lie à la protéine cible.
PCT/EP2006/012434 2005-12-23 2006-12-22 Procede de production d'une proteine chimiquement modifiee WO2007076974A2 (fr)

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DE200510061934 DE102005061934A1 (de) 2005-12-23 2005-12-23 Verfahren zur Herstellung eines chemisch modifizierten Proteins

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140317A2 (fr) * 2013-03-15 2014-09-18 Nbe-Therapeutics Llc Procédé de production d'un conjugué immunoligand/charge
US10188745B2 (en) 2014-12-23 2019-01-29 Nbe-Therapeutics Ag Binding protein drug conjugates comprising anthracycline derivatives

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BASHTOVYY DENYS ET AL: "Constrained modeling of spin-labeled major coat protein mutants from M13 bacteriophage in a phospholipid bilayer" PROTEIN SCIENCE, Bd. 10, Nr. 5, Mai 2001 (2001-05), Seiten 979-987, XP002450475 ISSN: 0961-8368 *
ERNST L A ET AL: "Cyanine Dye Labeling Reagents for Sulfhydryl Groups" CYTOMETRY, ALAN LISS, NEW YORK, US, Bd. 10, Nr. 1, 1989, Seiten 3-10, XP002249385 ISSN: 0196-4763 *
GIRIAT IZABELA ET AL: "Protein semi-synthesis in living cells." JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Bd. 125, Nr. 24, 18. Juni 2003 (2003-06-18), Seiten 7180-7181, XP002450472 ISSN: 0002-7863 *
GOODY ROGER S ET AL: "Combining chemical and biological techniques to produce modified proteins." CHEMBIOCHEM : A EUROPEAN JOURNAL OF CHEMICAL BIOLOGY 3 MAY 2002, Bd. 3, Nr. 5, 3. Mai 2002 (2002-05-03), Seiten 399-403, XP002450476 ISSN: 1439-4227 *
HAHN M E ET AL: "Manipulating proteins with chemistry: a cross-section of chemical biology" TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER, HAYWARDS, GB, Bd. 30, Nr. 1, Januar 2005 (2005-01), Seiten 26-34, XP004710938 ISSN: 0968-0004 *
STRATIKOS EFSTRATIOS ET AL: "Mapping the serpin-proteinase complex using single cysteine variants of alpha1-proteinase inhibitor Pittsburgh" JOURNAL OF BIOLOGICAL CHEMISTRY, Bd. 273, Nr. 25, 19. Juni 1998 (1998-06-19), Seiten 15582-15589, XP002450474 ISSN: 0021-9258 *
ZUGER SARA ET AL: "Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies" NATURE BIOTECHNOLOGY, Bd. 23, Nr. 6, Juni 2005 (2005-06), Seiten 736-740, XP002450473 ISSN: 1087-0156 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014140317A2 (fr) * 2013-03-15 2014-09-18 Nbe-Therapeutics Llc Procédé de production d'un conjugué immunoligand/charge
WO2014140317A3 (fr) * 2013-03-15 2014-12-24 Nbe-Therapeutics Llc Procédé de production d'un conjugué immunoligand/charge
US9872923B2 (en) 2013-03-15 2018-01-23 Nbe Therapeutics Ag Method of producing an immunoligand/payload conjugate
US10864277B2 (en) 2013-03-15 2020-12-15 Nbe Therapeutics Ag Method of producing an immunoligand/payload conjugate
US11364301B2 (en) 2013-03-15 2022-06-21 Nbe-Therapeutics Ag Method of producing an immunoligand/payload conjugate
US11986535B2 (en) 2013-03-15 2024-05-21 Nbe Therapeutics Ag Method of producing an immunoligand/payload conjugate
US10188745B2 (en) 2014-12-23 2019-01-29 Nbe-Therapeutics Ag Binding protein drug conjugates comprising anthracycline derivatives
US10517959B2 (en) 2014-12-23 2019-12-31 Nbe-Therapeutics Ag Binding protein drug conjugates comprising anthracycline derivatives
US10960083B2 (en) 2014-12-23 2021-03-30 Nbe-Therapeutics Ag Binding protein drug conjugates comprising anthracycline derivatives
US11833120B2 (en) 2014-12-23 2023-12-05 Nbe-Therapeutics Ag Binding protein drug conjugates comprising anthracycline derivatives

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