WO2004076670A1 - Enhanced expression of fusion polypeptides with a biotinylation tag - Google Patents
Enhanced expression of fusion polypeptides with a biotinylation tag Download PDFInfo
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- WO2004076670A1 WO2004076670A1 PCT/EP2004/001973 EP2004001973W WO2004076670A1 WO 2004076670 A1 WO2004076670 A1 WO 2004076670A1 EP 2004001973 W EP2004001973 W EP 2004001973W WO 2004076670 A1 WO2004076670 A1 WO 2004076670A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
- C07K1/1077—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 residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to nucleic acids encoding a polypeptide capable of being biotinylated by holocarboxylase synthetase.
- the present invention relates to the formation of fusion polypeptides comprising an N-terminal polypeptide capable of being biotinylated by holocarboxylase synthetase and a C-terminal polypeptide with a biological function.
- the invention relates to the enhanced formation of such fusion polypeptides by means of expression in vitro or in vivo E.coli-based expression systems.
- the invention therefore relates to the field of molecular biology, but given the diverse uses for recombinant proteins, the invention also relates to the fields of chemistry, pharmacology, biotechnology, and medical diagnostics.
- the enzyme holocarboxylase synthetase of E.coli (BirA, a biotin ligase) catalyzes in vivo the biotinylation, that is the covalent addition of biotin to the ⁇ -amino group of a lysine side chain in its natural substrate, biotin carboxyl carrier protein (BCCP) (Cronan, J.E., Jr., et al., J. Biol. Chem. 265 (1990) 10327-10333).
- BCCP biotin carboxyl carrier protein
- This protein is a subunit of acetyl-CoA carboxylase.
- the reaction is catalysed by the biotin- protein ligase, the product of the BirA gene (Cronan, J.E., Jr., Cell 58 (1989) 427-429).
- a BirA substrate consisting of a sequence of 13 amino acids was defined as a biotinylation polypeptide in fusion polypeptides (Schatz, P.J., Biotechnology 11 (1993) 1138-1143).
- WO 95/04069 describes biotinylation peptides that can be fused to other peptides or proteins of interest using recombinant DNA techniques. The resulting fusion polypeptides can be biotinylated in vivo or in vitro by BirA holocarboxylase synthetase. Particularly WO 95/04069 describes the expression of such fusion polypeptides in E.coli and anticipates expression in cell-free expression systems. But both documents are completely silent regarding the impact of the nucleic acid sequence that is encoding an N-terminal biotinylation polypeptide on the expressed quantity of the fusion polypeptide.
- US 5,723,584, US 5,874,239, US 5,932,433 and US 6,265,552 provide further amino acid sequences for biotinylation polypeptides to be used for generating fusions with polypeptides of interest.
- N-terminally tagged fusion polypeptides the documents describe the chemical synthesis of nucleic acid sequences that were biased in order to fit a consensus biotinylation polypeptide sequence. However, the documents are completely silent regarding the impact of the nucleic acid sequence that is encoding an N- terminal biotinylation polypeptide on the expressed quantity of the fusion polypeptide.
- the biotinylation polypeptide used in the present invention (SEQ ID NO: 1, AviTagTM) is comprised in the pAN-4, pAN-5, pAN-6 series of expression vectors distributed by Avidity Inc., Denver, Colorado, USA.
- the set of 3 different pAN vectors are designed for cloning and expression of N-terminal tagged fusion polypeptides in each reading frame.
- the DNA sequence encoding the biotinylation polypeptide is the DNA sequence of SEQ ID NO: 3.
- E.coli-based cellular expression systems are well-known to the art and include US 5,232,840 regarding an optimized ribosome-binding site. Particularly cellular E.coli expression systems using the T7 promoter are described in US 4,952,496, US 5,693,489 and US 5,869,320.
- Codon usage is one of the best known parameters impacting on the expressed quantity of a polypeptide. Genes in both prokaryotes and eukaryotes show a non-random usage of synonymous codons.
- the systematic analysis of codon usage patterns in E. coli led to the following observations (de Boer, H.A., and Kastelein, R.A., In: Maximizing gene expression, Reznikoff, W.S., and Gold, L., (eds.), Butterworfhs, Boston, 1986, pp. 225- 285): (1) There is a bias for one or two codons for almost all degenerate codon families. (2) Certain codons are most frequently used by all different genes irrespective of the abundance of the protein.
- the problem to be solved is to provide the means to further enhance in a cell-free as well as in a cellular expression system the formation of a fusion polypeptide that comprises a biotinylation polypeptide.
- the invention provides the means to enhance in E.coli-based expression systems the formation of fusion polypeptides containing as an N-terminal tag a biotinylation polypeptide. It was surprisingly found that specifically exchanging in the nucleic acid sequence encoding the biotinylation polypeptide nucleotides at 11 discrete positions enhances the formation of the total fusion polypeptide by at least 40%.
- the invention provides nucleic acids encoding a polypeptide capable of being biotinylated by holocarboxylase synthetase.
- the invention provides an expression vector comprising a nucleic acid according to the invention.
- the invention provides a method of preparing a biotinylated polypeptide in a cell-free polypeptide synthesis reaction mixture.
- the invention provides use of a nucleic acid according to the invention for constructing, by way of genetic engineering, a nucleic acid encoding a fusion polypeptide and expressing the same, whereby the fusion polypeptide consists of an N-terminal polypeptide capable of being biotinylated by holocarboxylase synthetase, and a C- terminal polypeptide with a biological function.
- polypeptide with a biological function refers to a polypeptide which possesses a biological function or activity which is identified through a defined functional assay and which is associated with a particular biologic, morphologic, or phenotypic alteration in a cell or a virus.
- polypeptides with a biological function are receptors, transcription factors, kinases, polypeptide subunits of complexes, or antibodies.
- polypeptide with a biological function also encompasses "functional fragments" thereof, thus including all fragments of a the polypeptide with a biological function that retain an activity of the polypeptide.
- Functional fragments for example, can vary in size from a polypeptide fragment as small as, e.g., an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
- polypeptide with a biological function encompasses a hybrid polypeptide, that is to say a fusion of two or more polypeptides with biological functions.
- polypeptide denotes a polymer composed of amino acid monomers joined by peptide bonds.
- a “peptide bond” is a covalent bond between two amino acids in which the -amino group of one amino acid is bonded to the -carboxyl group of the other amino acid. All amino acid or polypeptide sequences, unless otherwise designated, are written from the amino terminus (N-terminus) to the carboxy terminus (C-terminus). Amino acid identification uses the three-letter abbreviations as well as the single-letter alphabet of amino acids, i.e.
- Asp D Aspartic acid He 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, Gin Q Glutamine, Met M Methionine, Asn N Asparagine.
- biotinylation polypeptide is a "polypeptide capable of being biotinylated by holocarboxylase synthetase".
- the amino acid sequence of the biotinylation polypeptide provides a sequence motif containing an acceptor site for "biotinylation", that is the covalent attachment of a biotin molecule by holocarboxylase synthetase.
- the term "tagging" or “tagging a target sequence” refers to introducing by recombinant methods a nucleic acid encoding a "tag” such as a biotinylation polypeptide into a polypeptide-encoding nucleic acid, i.e. a "target sequence” so that the recombinant nucleic acid encodes a fusion polypeptide which comprises the tag at its C- or N-terminus.
- fusion polypeptide refers to a polypeptide which has been tagged, e.g. with a biotinylation polypeptide.
- the amino acid sequence of a fusion polypeptide may comprise the amino acid sequence of the biotinylation polypeptide and the amino acid sequence of a target polypeptide.
- the target polypeptide itself is a polypeptide with a biological function.
- Nucleic acid refers to DNA or RNA which may be single- or double- stranded, and represents the sense strand when single-stranded. Nucleic acids are polymers with nucleotides as monomers. Nucleotides are composed of a phosphate moiety, a sugar moiety (ribose or deoxyribose) and an aglyconic heterocyclic moiety, the so-called nucleobase. In a nucleic acid sequence a single letter defines a nucleotide by its nucleobase, i.e. adenine (A), guanine (G), cytosine (C) and thymine (T) or uracil (U).
- A adenine
- G guanine
- C cytosine
- T thymine
- U uracil
- Nucleic acids encoding fusion polypeptides can be prepared by chemical methods or by genetic engineering.
- a fusion polypeptide can be obtained by means of "expression" of a nucleic acid encoding the same, that is as a result of transcription and translation of the nucleic acid.
- a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid.
- a nucleic acid encoding a biotinylation polypeptide is operably linked to a nucleic acid encoding a polypeptide with a biological function if it results in the expression of a fusion polypeptide capable of being biotinylated;
- a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
- operably linked means that the nucleic acids being linked are contiguous and, in the case of a nucleic acid encoding, e.g., a biotinylation polypeptide, contiguous and in reading phase.
- a nucleic acid encoding e.g., a biotinylation polypeptide
- linking is accomplished by ligation at convenient restriction sites. If such sites do not exist then synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice.
- nucleic acid sequences are written in the direction from the 5' (stands for prime) end to the 3' end also referred to as 5' to 3'.
- the nucleic acid sequences of the invention that encode a polypeptide of SEQ ID NO: 1 are different from previously published nucleic acid sequences such as SEQ ID NO: 3 because of the degeneracy of the genetic code and encode the same polypeptide.
- Degenerate code stands for a genetic code in which a particular amino acid can be coded by two or more different codons. Degeneracy occurs because of the fact that of the 64 possible base triplets, 3 are used to code the stop signals, and the other 61 are left to code for only 20 different amino acids.
- expression system is well understood in the art to mean either an in vitro system or a cellular or multicellular organism capable of translating or transcribing and translating nucleotide sequences to produce polypeptides.
- An example for an in vitro expression system that is to say a cell-free polypeptide synthesis reaction mixture, is described in Zubay, G., Annu. Rev. Genet. 7 (1973) 267-287. Spirin et al. developed in 1988 a continuous-flow cell-free translation and coupled transcription/translation system in which a relatively high amount of protein synthesis occurs (Spirin, A.S., et al., Science 242 (1988) 1162-1164).
- the invention provides a nucleic acid of SEQ ID NO: 2 encoding a polypeptide of SEQ ID NO: 1 capable of being biotinylated by holocarboxylase synthetase, characterized in that said nucleic acid differs from SEQ ID NO: 3 by nucleotide exchanges at 6 or more positions selected from the group consisting of the positions 4, 5, 6, 9, 10, 12, 15, 18, 21, 24 or 30, and said nucleic acid, as compared to SEQ ID NO: 3, enhances the formation of a fusion polypeptide, consisting of an N-terminal polypeptide according to SEQ ID NO: 1 and a C-terminal polypeptide with a biological function, by means of expression from a nucleic acid encoding said fusion polypeptide in a cell-free polypeptide synthesis reaction mixture in that at least 40% more fusion polypeptide is formed, whereby the nucleic acid encoding said fusion polypeptide consists of a nucleic acid encoding said N
- the nucleic acid that is containing A or T at position 4, C or G at position 5, C or T at position 6, A, C or T at position 9, C or T at position 10, A or G at position 12, C or T at position 15, C or T at position 18, C or T at position 21, C or T at position 24, and A or T at position 30, is characterized in that between 5 and 11 nucleotides at said positions are identical to the nucleotides at the same positions in SEQ ID NO: 4, with the proviso that all nucleotides at said positions are identical to the nucleotides at the same positions in SEQ ID NO: 3 or SEQ ID NO: 4, or 10 nucleotides at said positions except position 9 are identical to the nucleotides at the same positions in SEQ ID NO: 4 or SEQ ID NO: 3, and the nucleotide at position 9 is T.
- the nucleic acid is characterized in that the nucleic acid is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
- Another aspect of the invention is an expression vector comprising a nucleic acid according to the invention.
- Yet another aspect of the invention is a method of preparing a biotinylated polypeptide in a cell-free polypeptide synthesis reaction mixture which contains an RNA polymerase, ribosomes, RNA, ATP, GTP, nucleotides and amino acids, comprising the steps of (a) forming in said reaction mixture a fusion polypeptide, consisting of an N-terminal polypeptide according to SEQ ID NO: 1 and a C-terminal polypeptide with a biological function, by means of expression from a nucleic acid consisting of a nucleic acid according to any of the claims 1 to 3 operably linked to a nucleic acid encoding the C-terminal polypeptide; (b) biotinylating said fusion polypeptide in the presence of biotin and holocarboxylase synthetase; (c) isolating said biotinylated fusion polypeptide from said mixture; or incubating said mixture with immobilized avidin or streptavi
- RNA polymerase is a DNA-dependent RNA polymerase.
- a very much preferred RNA polymerase is T7 RNA polymerase.
- Holocarboxylase synthetase (EC6.3.4.15, biotin protein ligase, BirA) is an enzyme that catalyses in E.coli the covalent attachment of biotin to its natural substrate, that is BCCP.
- Biotin ligase is highly specific and reacts only on biotinylation polypeptides showing a very high degree of conservation in the primary structure of the biotin attachment domain. This domain includes preferably the highly conserved AMKM tetrapeptide (Chapman-Smith, A., and Cronan, J.E., Jr., J. Nutr. 129, 2S Suppl., (1999) 477S-484S).
- Recombinant BirA enzyme is described in WO 99/37785.
- holocarboxylase synthetase can be added to an in vitro expression system as an active enzyme or can be added as a nucleic acid (in an expression vector, e.g. RNA, DNA) which is expressed (transcribed/translated) in the system like the fusion polypeptide.
- an expression vector e.g. RNA, DNA
- the method is characterized in that the reaction mixture contains a nucleic acid encoding holocarboxylase synthetase according to SEQ ID NO: 25 that is expressed in said reaction mixture to provide holocarboxylase synthetase polypeptide.
- an active enzyme it is used preferably in an amount of about 10,000 to 15,000 units, preferably 12,500 units.
- a preferred active enzyme (EC6.3.4.15) is supplied by Avidity Inc. (Denver, Colorado, USA).
- the method is characterized in that the reaction mixture contains a nucleic acid encoding holocarboxylase synthetase according to SEQ ID NO: 25 that is expressed in the reaction mixture to provide holocarboxylase synthetase polypeptide.
- the amount of nucleic acid depends on the expression rate of the used vector and the necessary amount of BirA enzyme in the reaction mixture. 1 ng of BirA plasmid DNA (e.g.
- E.coli expression vector such as pIVEX vectors, supplied by Roche Diagnostics GmbH, Mannheim, Germany; http://www.biochem.roche.com/RTS), or even less, is sufficient for a quantitative biotinylation reaction of the tagged fusion polypeptides.
- the maximum yield of expressed and specifically biotinylated fusion polypeptide is achieved, when the desired fusion polypeptide-encoding plasmid DNA is added at 10-15 ⁇ g and the plasmid DNA, being responsible for the coexpression of BirA, is introduced with an amount between 1-10 ng.
- the ratio of fusion polypeptide-encoding plasmid DNA to BirA-encoding plasmid DNA was found to be optimal at a ratio of about 1500:1. It was found that the same level as above is sufficient for quantitative biotinylation of the expressed fusion protein.
- D(+) -biotin was added at 1 to 10 ⁇ M, preferably in about 2 ⁇ M to the reaction mixture.
- biotinylation occurs under standard reaction conditions, preferably within 10 to 30 hours at 20°C to 36°C, most preferably at about 30°C, and the reaction mixture is preferably, after dialysis, for concentration and buffer exchange, centrifuged.
- the solution is, due to its high purity, directiy used for immobilization of the fusion polypeptide on surfaces which contain immobilized avidin or streptavidin (e.g. microtiter plates or biosensors) without further purification.
- immobilized avidin or streptavidin e.g. microtiter plates or biosensors
- biotinylated polypeptides produced according to the present invention can be purified further under native conditions using matrices containing immobilized (preferably monomeric) avidin, streptavidin, or derivatives thereof.
- immobilized (preferably monomeric) avidin, streptavidin, or derivatives thereof A variety of useful physically (Kohanski, R.A., and Lane, M.D., Methods Enzymol. 184 (1990) 194-200), chemically (Morag, E., et al., Anal. Biochem. 243 (1996) 257-263) and genetically (Sano, T., and Cantor, C.R., Proc. Natl. Acad. Sci.USA 92 (1995) 3180-3184) modified forms of avidin or streptavidin have been described that still bind biotin specifically but with weaker affinity to facilitate a one step purification procedure.
- Yet another aspect of the invention is the use of a nucleic acid according to the invention for constructing, by way of genetic engineering, a nucleic acid encoding a fusion polypeptide, whereby the fusion polypeptide consists of an N-terminal polypeptide of SEQ ID NO: 1 and a C-terminal polypeptide with a biological function.
- Methods for constructing by way of genetic engineering are well known to the art and are described, in e.g. Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 3rd edition, CSHL Press, 2001.
- Yet another aspect of the invention is the use of a nucleic acid according to the invention for expressing a fusion polypeptide, whereby the fusion polypeptide consists of an N- terminal polypeptide of SEQ ID NO: 1 and a C-terminal polypeptide with a biological function.
- a preferred embodiment of the invention is the use characterized in that the fusion polypeptide is expressed in a cell-free polypeptide synthesis reaction mixture.
- a preferred cell-free polypeptide synthesis reaction mixture is the RTS500 in vitro expression system supplied by Roche Diagnostics GmbH (Mannheim, Germany; catalogue number 3246817).
- a preferred E.coli strain is a BL21 (DE 3) strain. Even more preferred is a BL21 (DE 3) Lys S strain. These strains express an active T7 RNA polymerase.
- Such a strain can be used to transcribe a gene carried by an expression vector, whereby the vector comprises, e.g., a nucleic acid encoding a fusion polypeptide that is operably linked to the T7 promoter.
- Examples for vectors that have incorporated the T7 promoter and that are capable of being transcribed in the BL21 (DE 3) strain or the BL21 (DE 3) Lys S strain of E.coli are pET vectors (Novagen Inc., Madison, Wisconsin, USA) or pIVEX vectors (Roche Diagnostics GmbH, Mannheim, Germany). Methods for expressing fusion polypeptides are well known to the art and are described (e.g. in: Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 3rd edition, CSHL Press, 2001. Also in: Gu, J., et al., Biotechniques 17 (1994) 257, 260, 262).
- Figure 1A Coomassie-stained SDS gel.
- the numbers on the bottom indicate the numbers of the SDS gel lanes.
- the numbers on the left hand side of the gel indicate molecular weight (given in [kDa]) as indicated by the molecular weight markers to the left of lane 1.
- In vitro expression see
- Example 3 of fusion polypeptides from pIVEX-2.8 CAT WT AviTagTM with the wildtype sequence encoding the N-terminal tag (lane 1, 5), pIVEX-2.8 CAT mut AviTagTM with the sequence of SEQ ID NO: 12 encoding the N-terminal tag (lane 2, 6), pIVEX-2.8 EPO WT AviTagTM with the wildtype sequence encoding the N-terminal tag (lane 3, 7), pIVEX-2.8 EPO mut AviTagTM with the sequence of SEQ ID NO: 12 encoding the N-terminal tag (lane 4, 8).
- the total protein suspension of each cell-free polypeptide synthesis reaction mixture was applied in lanes 1-4, the pellet fraction in lanes 5-8.
- Figure IB Densitometric analysis as described in Example 4 was performed on the areas indicated. The numbers on the bottom indicate the numbers of the SDS gel lanes as in Figure 1A. It is noted that for the lanes 7 and 8 the numbering of densitometrically quantified bands is changed. Thus, the band designated with "8" is in lane 7 and the band designated with "9” is in lane 8. The values obtained from densitometric quantification are given in Table 1 (Example 4) and are tabulated with reference to the numbering of SDS gel lanes.
- Figure 2 pIVEX-GFP WT AviTagTM
- Figure 3 pIVEX-2.8 CAT mut AviTagTM; the site denoted "Xa factor” indicates a cleavage site for factor Xa protease.
- Figure 4 pIVEX-2.8 EPO mut AviTagTM; the site denoted "Xa factor” indicates a cleavage site for factor Xa protease.
- the AviTagTM biotinylation polypeptide comprises a sequence of 15-17 amino acid residues and can be used as a tag in fusion polypeptides.
- the AviTagTM is capable of being biotinylated at a lysine residue by a biotin protein ligase such as the polypeptide encoded by the E.coli BirA gene (Murtif, V.L., and Samols, D., J. Biol. Chem. 262 (1987) 11813-11816).
- the AviTagTM biotinylation polypeptide used for the present invention is represented by SEQ ID NO: 1.
- a DNA sequence encoding the AviTagTM and expression vectors in which the DNA sequence is incorporated are commercially available from Avidity Inc. (Denver, Colorado, USA).
- the original DNA sequence of which variants were generated is SEQ ID NO: 3. This sequence is also referred to as "wildtype sequence” or "wildtype DNA sequence”.
- the wildtype DNA sequence was placed in-frame in front of the test protein green fluorescent protein (GFP; Crameri., A., et al., Nat. Biotechnol. 14 (1996) 315-319) by using conventional cloning methods (Sambrook, Fritsch & Maniatis, Molecular Cloning, A Laboratory Manual, 3rd edition, CSHL Press, 2001).
- GFP green fluorescent protein
- To create mutant sequences of the first ten codons of the wildtype sequence the following two sets of degenerated oligonucleotides were synthesized.
- the mutated sequences that were synthesized exploited the codon usage for each amino acid without changing the primary sequence.
- N any base
- Y pyrimidine (C or T)
- R purine (G or A)
- H not G (i.e. A, T or C).
- a PCR reaction was made with the pIVEX- GFP WT AviTagTM (SEQ ID NO: 29) vector as template.
- the restriction enzymes Xbal and Ncol the PCR products were cleaved, firstly at the Xbal site in the forward primer and secondly at the Ncol site in the reverse primer.
- the pIVEX-GFP WT AviTagTM vector was cleaved with the same restriction enzymes and the vector fragment was isolated. Subsequently, the cleaved fragments were inserted into the pIVEX-GFP AviTagTM vector fragments.
- the plasmids were ligated and subsequently transformed into a BL21 (DE 3) Lys S strain of E.coli (Novagen Inc., Madison, Wisconsin, USA) and plated out on LB medium with ampicillin (100 ⁇ g/ml), chloramphenicol (100 ⁇ g/ml) and IPTG (0.2 mM). After one day of growth bacterial colonies were screened under UV light for GFP expression. The colonies with the brightest fluorescence as judged by visual inspection were picked and plasmids from these colonies were isolated. The AviTagTM-encoding DNA of these plasmids was subjected to sequence analysis.
- the screening procedure resulted in a number of mutant variants of the wildtype sequence encoding the AviTagTM, whereby these variants stimulated a visibly increased GFP signal as compared to the signal of control transformants expressing the pIVEX-GFP WT AviTagTM vector.
- mutant variants of the wildtype sequence i.e. DNA sequences encoding a polypeptide of SEQ ID NO: 1 capable of being biotinylated by holocarboxylase synthetase, are represented in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.
- the wildtype sequence was compared with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, and a consensus sequence was derived for the mutant variants.
- the consensus DNA sequence encoding SEQ ID NO: 1 was found to differ from the wildtype sequence, that is the sequence according to SEQ ID NO: 3, by nucleotide exchanges at 6 or more positions selected from the group consisting of the positions 4, 5, 6, 9, 10, 12, 15, 18, 21, 24 or 30.
- consensus DNA sequence was found to contain A or T at position 4, C or G at position 5, C or T at position 6, A, C or T at position 9, C or T at position 10, A or G at position 12, C or T at position 15, C or T at position 18, C or T at position 21, C or T at position 24, and A or T at position 30.
- the consensus sequence is given in SEQ ID NO: 2.
- nucleotides at said positions were found to be identical to the nucleotides at the same positions in SEQ ID NO: 4, with the proviso that all nucleotides at said positions were found to be identical to the nucleotides at the same positions in SEQ ID NO: 3 or SEQ ID NO: 4, or 10 nucleotides at said positions except position 9 were found to be identical to the nucleotides at the same positions in SEQ ID NO: 4 or SEQ ID NO: 3, and the nucleotide at position 9 was then found to be T.
- the mutated AviTagTM sequence according to SEQ ID NO: 12 was inserted in-frame in front of the chloramphenicol acetyl transferase (CAT) gene and the erythiOpoietin (EPO) gene by way of a PCR cloning approach analogous to the approach described in Example 1.
- CAT chloramphenicol acetyl transferase
- EPO erythiOpoietin
- control plasmids pIVEX-2.8 CAT WT AviTagTM and pIVEX-2.8 EPO WT AviTagTM were generated that differed from pIVEX-2.8 CAT mut AviTagTM and pIVEX-2.8 EPO mut AviTagTM in that the wildtype AviTagTM sequence, i.e. SEQ ID NO: 3 replaced SEQ ID NO: 12.
- All four of these plasmids were then used for a polypeptide synthesis reaction using the RTS 500 HY Kit (Roche Diagnostics GmbH, Mannheim, Germany) as an in-vitro expression system. Each plasmid was used for a separate in-vitro expression.
- the polypeptide synthesis reactions were performed identically and in line with the instructions of the supplier. After the reactions were ended, 0.5 ⁇ l aliquots of each reaction mixture were directly applied on an SDS-PAGE gel. Another aliquot of each reaction was centrifuged for 15 min at 30,000 x g. The supernatants were removed and the pellet fractions were resuspended in the original volume in SDS sample buffer. Again 0.5 ⁇ l were applied on the same SDS Page gel.
- Figure 1 shows the result.
- the fusion polypeptides encoded by the wildtype AviTagTM DNA sequence that was operably linked to the coding sequences of either CAT or EPO were present in smaller quantities as opposed to those fusion polypeptides in which the N-terminal tag was encoded by the mutated sequence of SEQ ID NO: 12.
- EPO in its unglycosylated form can be detected primarily in the pellet fraction.
- the amounts of expressed fusion polypeptides were quantified by way of densitometric measurements of coomassie-stained bands in SDS gels that were obtained using the Lumi Imager Fl and the LumiAnalyst Software (Roche Diagnostics GmbH, Mannheim, Germany). Measurements were made according to the instructions of the manufacturer. Each analysed each gel contained control lanes in which defined amounts of marker proteins were electrophoresed in order to provide reference points for quantification. Table 1 provides results from the parallel experiments described in Example 3 and Figure 1.
- mutant variant of the wildtype sequence as given in SEQ ID NO: 12 enhances the formation of the fusion polypeptide, consisting of an N-terminal polypeptide according to SEQ ID NO: 1 and a C-terminal polypeptide with a biological function in that at least 40% more fusion polypeptide is formed.
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EP04715289A EP1599589A1 (en) | 2003-02-28 | 2004-02-27 | Enhanced expression of fusion polypeptides with a biotinylation tag |
CA002513742A CA2513742A1 (en) | 2003-02-28 | 2004-02-27 | Enhanced expression of fusion polypeptides with a biotinylation tag |
US11/211,339 US20060046285A1 (en) | 2003-02-28 | 2005-08-25 | Enhanced expression of fusion polypeptides with a biotinylation tag |
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EP03004326A EP1452601A1 (en) | 2003-02-28 | 2003-02-28 | Enhanced expression of fusion polypeptides with a biotinylation tag |
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EP (2) | EP1452601A1 (en) |
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US7795411B2 (en) * | 2006-10-05 | 2010-09-14 | Fred Hutchinson Cancer Research Center | Vectors for expressing in vivo biotinylated recombinant proteins |
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WO2012100343A1 (en) | 2011-01-28 | 2012-08-02 | National Research Council Of Canada | Engineering of immunoglobulin domains |
US9066986B2 (en) | 2010-10-01 | 2015-06-30 | National Research Council Of Canada | Anti-CEACAM6 antibodies and uses thereof |
WO2015131257A1 (en) | 2014-03-06 | 2015-09-11 | National Research Council Of Canada | Insulin-like growth factor 1 receptor -specific antibodies and uses thereof |
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WO2016199097A1 (en) | 2015-06-10 | 2016-12-15 | National Research Council Of Canada | Carbonic anhydrase ix-specific antibodies and uses thereof |
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WO2018109663A1 (en) | 2016-12-12 | 2018-06-21 | National Research Council Of Canada | Antibody variants transmigrating the blood-brain barrier and uses thereof |
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US10736955B2 (en) | 2017-11-23 | 2020-08-11 | Theraphage Inc. | Fusion protein comprising bacteriophage λ GpD fused to a GP2 antigen derived from HER-2/neu |
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Also Published As
Publication number | Publication date |
---|---|
EP1599589A1 (en) | 2005-11-30 |
CA2513742A1 (en) | 2004-09-10 |
US20060046285A1 (en) | 2006-03-02 |
EP1452601A1 (en) | 2004-09-01 |
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