THIOL-BASED DEEP EUTECTIC SOLVENT

Abstract

A deep eutectic solvent consisting of (2-hydroxyethyl) trimethyl ammonium chloride and dithiothreitol in a molar ratio of from 1:2 to 1:3 and from 0% to 10% co-solvent, and methods of enzymatic production of polypeptides using the deep eutectic solvent.

Claims

1. A deep eutectic solvent consisting of (2-hydroxyethyl) trimethyl ammonium chloride and dithiothreitol in a molar ratio of from 1:2 to 1:3 and from more than 0% to 10% of co-solvent.

2. A deep eutectic solvent consisting of a) i) (2-hydroxyethyl) trimethyl ammonium chloride, and ii) 4-mercapto-1-butanol or 1-mercapto-2-propanol in a molar ratio of i) to ii) of about 1:2, and b) of from more than 0% to about 10% co-solvent.

3. A deep eutectic solvent consisting of (2-hydroxyethyl) trimethyl ammonium chloride and sodium 2-mercaptoethansolfonate in a molar ratio of about 1:1 and of from more than 0% to about 10% co-solvent.

4. The deep eutectic solvent of claim 1, wherein the co-solvent is an aqueous co-solvent.

5. The deep eutectic solvent of claim 4, comprising from more than 0% up to about 5% (v/v) aqueous co-solvent.

Description

DESCRIPTION OF THE FIGURES

[0232] FIG. 1 Chromatogram of the reaction product of Example 4; 8.433 min=GGG-biotin, 13.350 min=Sa-SrtA, 20.942=LCR640-LPETG (educt), 21.987 min=LCR640-LPETG-biotin (product).

[0233] The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

EXAMPLES

Recombinant DNA Techniques

[0234] Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions.

Gene and Oligonucleotide Synthesis

[0235] Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

Description of the Basic/Standard Mammalian Expression Plasmid

[0236] For the expression of a desired gene/protein (e.g. full length antibody heavy chain, full length antibody light chain, or an Fc-chain containing an oligoglycine at its N-terminus) a transcription unit comprising the following functional elements is used: [0237] the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, [0238] a human heavy chain immunoglobulin 5-untranslated region (5UTR), [0239] a murine immunoglobulin heavy chain signal sequence, [0240] a gene/protein to be expressed (e.g. shortened Sortase A of Staphylococcus aureus), and [0241] the bovine growth hormone polyadenylation sequence (BGH pA).

[0242] Beside the expression unit/cassette including the desired gene to be expressed the basic/standard mammalian expression plasmid contains [0243] an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and [0244] a beta-lactamase gene which confers ampicillin resistance in E. coli.

Protein Determination

[0245] The protein concentration of purified polypeptides was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence of the polypeptide.

Example 1

[0246] Generation of an expression plasmid for soluble sortase A

Staphylococcus aureus Derived Sortase A

[0247] The sortase gene encodes an N-terminally truncated Staphylococcus aureus sortase A (60-206) molecule (amino acid sequence of SEQ ID NO: 05).

[0248] The expression plasmid for the transient expression of soluble sortase in HEK293 cells comprised besides the soluble sortase expression cassette an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.

[0249] The transcription unit of the soluble sortase comprised the following functional elements: [0250] the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, [0251] a human heavy chain immunoglobulin 5-untranslated region (5UTR), [0252] a murine immunoglobulin heavy chain signal sequence, [0253] a purification tag encoding nucleic acid, [0254] an N-terminally truncated S. aureus sortase A encoding nucleic acid, and [0255] the bovine growth hormone polyadenylation sequence (BGH pA).

[0256] The amino acid sequence of the mature soluble sortase is

TABLE-US-00009 (SEQIDNO:05) QAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENES LDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIR DVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVK.

[0257] The purification tag has the amino acid sequence MRGSHHHHHHGS (SEQ ID NO: 32).

Staphylococcus pyogenes Derived Sortase A

[0258] The sortase gene encodes an N-terminally truncated Staphylococcus pyogenes sortase A molecule (amino acid sequence of SEQ ID NO: 156).

[0259] The expression plasmid for the transient expression of soluble sortase in HEK293 cells comprised besides the soluble sortase expression cassette an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.

[0260] The transcription unit of the soluble sortase comprised the following functional elements: [0261] the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, [0262] a human heavy chain immunoglobulin 5-untranslated region (5UTR), [0263] a murine immunoglobulin heavy chain signal sequence, [0264] a purification tag encoding nucleic acid, [0265] an N-terminally truncated S. pyogenes sortase A encoding nucleic acid, and [0266] the bovine growth hormone polyadenylation sequence (BGH pA).

[0267] The amino acid sequence of the mature soluble sortase is

TABLE-US-00010 (SEQIDNO:156) VLQAQMAAQQLPVIGGIAIPELGINLPIFKGLGNTELIYGAGTMKEEQVM GGENNYSLASHHIFGITGSSQMLFSPLERAQNGMSIYLTDKEKIYEYIIK DVFTVAPERVDVIDDTAGLKEVTLVTCTDIEATERIIVKGELKTEYDFDK APADVLKAFNHSYNQVST.

[0268] The purification tag has the amino acid sequence MRGSHHHHHHGS (SEQ ID NO: 32).

Listeria monocytogenes Derived Sortase A

[0269] The sortase gene encodes an N-terminally truncated Listeria monocytogenes sortase A (73-222) molecule (amino acid sequence of SEQ ID NO: 06).

[0270] The expression plasmid for the transient expression of soluble sortase in HEK293 cells comprised besides the soluble sortase expression cassette an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.

[0271] The transcription unit of the soluble sortase comprised the following functional elements: [0272] the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, [0273] a human heavy chain immunoglobulin 5-untranslated region (5UTR), [0274] a murine immunoglobulin heavy chain signal sequence, [0275] a purification tag encoding nucleic acid, [0276] an N-terminally truncated L. monocytogenes sortase A encoding nucleic acid, and [0277] the bovine growth hormone polyadenylation sequence (BGH pA).

[0278] The purification tag has the amino acid sequence MRGSHHHHHHGS (SEQ ID NO: 32).

Example 2

[0279] Transient expression and analytical characterization

[0280] The recombinant production was performed by transient transfection of HEK293 cells (human embryonic kidney cell line 293-derived) cultivated in F17 Medium (Invitrogen Corp.). For transfection 293-Fectin Transfection Reagent (Invitrogen) was used. Transfection was performed as specified in the manufacturer's instructions. Cell culture supernatants were harvested three to seven (3-7) days after transfection. Supernatants were stored at reduced temperature (e.g. 80 C.).

[0281] General information regarding the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

[0282] The protein concentration was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity was analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie brilliant blue.

Example 3

[0283] Production of thiol-based deep eutectic solvents

Dithiothreitol and Choline Chloride

[0284] Choline chloride and dithiothreitol were mixed in a 1:2.5 molar ratio. Choline chloride (0.1 mol) was employed as a dry powder and were added to 0.25 mol of dithiothreitol. The mixture was heated slowly over a flame and shaken until a clear, uniform solution had been formed.

[0285] The liquid was allowed to cool to room temperature. The yield was quantitative and the product had a melting point of lower than room temperature. About 10 g of the synthesized deep eutectic solvent were put in a closed vial and kept in a dry space.

Beta-mercaptoethanol and Choline Chloride

[0286] Choline chloride and beta-mercaptoethanol were mixed in a 1:2 molar ratio. Choline chloride (0.1 mol) was employed as a dry powder and was added to 0.2 mol of beta-mercaptoethanol. The mixture comprising was heated slowly over a flame and shaken until a uniform and clear solution had been formed.

[0287] The liquid was allowed to cool to room temperature. The yield was quantitative and the product had a melting point of lower than room temperature. About 10 g of the synthesized deep eutectic solvent were put in a closed vial and kept in a dry space.

[0288] 4-Mercapto-1-butanol and Choline Chloride

[0289] Choline chloride and 4-mercapto-1-butanol were mixed in a 1:2 molar ratio. Choline chloride (0.1 mol) was employed as a dry powder and was added to 0.2 mol of 4-mercapto-1-butanol. The mixture was heated slowly over a flame and shaken until a uniform solution was formed.

[0290] The liquid was allowed to cool to room temperature. The yield was quantitative and the product had a melting point of lower than room temperature. About 10 g of the synthesized deep eutectic solvent were put in a closed vial and kept in a dry space.

[0291] 1-Mercapto-2-propanol and Choline Chloride

[0292] Choline chloride and 1-mercapto-2-propanol were mixed in a 1:2 molar ratio. Choline chloride (0.1 mol) was employed as a dry powder and was added to 0.2 mol of 1-mercapto-2-propanol. The mixture was heated slowly over a flame and shaken until a uniform and clear solution was formed.

[0293] The liquid was allowed to cool to room temperature. The yield was quantitative and the product had a melting point of lower than room temperature. About 10 g of the synthesized deep eutectic solvent were put in a closed vial and kept in a dry space.

Sodium 2-mercaptoethansulfonate and Choline Chloride

[0294] Choline chloride and sodium 2-mercaptoethansulfonate were mixed in a 1:1 molar ratio. Choline chloride (0.1 mol) was employed as a dry powder and added to 0.1 mol of sodium 2-mercaptoethansulfonate. The mixture was heated slowly over a flame and shaken until a uniform solution had been formed.

[0295] The liquid was then centrifuged and the precipitate was discarded. About 2 g of the synthesized deep eutectic solvent were put in a closed vial and kept in a dry space.

[0296] 4-Mercaptophen Lacetic Acid and Choline Chloride

[0297] Choline chloride and 4-mercaptophenylacetic acid were mixed in a 1:2 molar ratio. Choline chloride (0.1 mol) was employed as a dry powder and was added to 0.2 mol of 4-mercaptophenylacetic acid. The mixture comprising was heated slowly over a flame and shaken. No eutecticum formed.

[0298] 2-Methyl-2-propanethiol and Choline Chloride

[0299] Choline chloride and 2-methyl-2-propanethiol were mixed in a 1:2 molar ratio. Choline chloride (0.1 mol) was employed as a dry powder and was added to 0.2 mol of 2-methyl-2-propanethiol. The mixture was heated slowly over a flame and shaken. No eutecticum formed.

[0300] 2-3-10-Mercaptopinane and Choline Chloride

[0301] Choline chloride and 2-,3-,10-mercaptopinane were mixed in a 1:2 molar ratio. Choline chloride (0.1 mol) employed as a dry powder and was added to 0.2 mol of 2-,3-,10-mercaptopinane. The mixture was heated slowly over a flame and shaken. No eutecticum formed

Example 4

[0302] Sortase mediated transamidation in thiol-based deep eutectic solvent

[0303] The molecules LCR640-ULPETGGRRC (LCR640 fluorophore conjugated to beta alanine (U); SEQ ID NO: 33) and GGG-biotin were dissolved in a thiol-based DES (choline chloride : dithiothreitol at molar ratio of 1:3) comprising 10% (v/v) water to a final concentration of 2 mM. In pure water LCR640-ULPETGGRRC solubilize to a concentration of less than 0.1 mM.

[0304] This solution was mixed with a solution comprising 1 mM Sa-SrtA (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM CaCl) at a volume ratio of 19:1. The reaction mixture was incubated for 5 h at 37 C.

[0305] The chromatogram of the reaction products is shown in FIG. 1. The retention times of the compounds are showing in the following Table.

TABLE-US-00011 retention time [min] compound 8.433 GGG-biotin 13.350 Sa-SrtA 20.942 LCR640-LPETG 21.987 LCR640-LPETG-biotin

[0306] The peak at 21.987 min. retention time corresponds to the reaction product.