WO2013178974A1 - Process for expression of crm197 - Google Patents
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- WO2013178974A1 WO2013178974A1 PCT/GB2013/000238 GB2013000238W WO2013178974A1 WO 2013178974 A1 WO2013178974 A1 WO 2013178974A1 GB 2013000238 W GB2013000238 W GB 2013000238W WO 2013178974 A1 WO2013178974 A1 WO 2013178974A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
- C12N15/72—Expression systems using regulatory sequences derived from the lac-operon
Definitions
- the present invention concerns a process for the expression of recombinant CRM 197 in E. coli.
- CRM 97 is a non-toxic mutant of diphtheria toxin having a single amino add substitution of glutamic acid for glycine.
- CRM197 is a well defined protein and functions as a carrier for polysaccharides and haptens making them immunogenic. It is utilized as a carrier protein in a number of approved conjugate vaccines for diseases such as meningitis and pneumococca) bacterial infections.
- WO2011/123139 discloses the expression of CRM197 in Pseudomonus fluorescens, but reports that expression of this protein in E coli hosts is confined to impractically low amounts.
- Stefan et al, Journal of Biotechnology 156 (2011 ) pp245-252 discloses the expression of CRM197 in E coli using a PET9a vector. Expression was only achieved with a synthetic gene bearing an N-terminal his-tag. Expression without the tag always failed.
- E. coli is a well characterised and commonly used host for the expression of recombinant proteins. Accordingly, it would be highly desirable to identify alternative methods of producing CRM 197 in E coli.
- a process for the intracellular expression of CRM197 in an E coli host comprising expressing a vector comprising a gene encoding CRM197 operably (inked to a promoter and at least one perfect palindrome operator sequence.
- Promoters which can be employed in the expression system of the present invention include phage RNA polymerase promoters and, commonly, E. coli RNA polymerase-based promoter systems.
- promoters which can be employed include T5, T7, T7A1 , T7A2, T7A3, ⁇ , pR, lac, lacUV5, trp, tac, trc, phoA and rrnB.
- a strong promoter is employed in the first aspect of the present invention. Strong promoters include promoters having a strength in P b i a -units, measured according to the method given by Bujard et al, The EMBO Journal, Vol 5, No. 11 , pp2987-2994 (1986) of greater than about 10, and preferably greater than about 15, such as in the range of from 20 to 40 or greater.
- Preferred promoters include T7A , T7A2, T7A3 and T5.
- Vectors and host cells comprising vectors for use in the process of first aspect also form part of the present invention.
- a process for the periplasmic expression of CRM 197 in an E coli host comprising expressing a vector comprising a gene encoding CRM197 and a secretion leader sequence operably linked to a weak promoter system comprising at least one perfect palindrome operator sequence.
- Weak promoter systems which can be employed in the second aspect of the present invention are those having a strength in P b i a -units, measured according to the method given by Bujard et al, The EMBO Journal, Vol 5, No. 11 , pp2987-2994 (1986) of less than about 10, and preferably less than about 7, such as in the range of from 1 to 6.
- Examples of promoters which can be employed in the expression system of the second aspect of the present invention include phage RNA polymerase promoters and, commonly, E. coli RNA polymerase-based promoter systems.
- the promoters employed may be promoters which have a strength in P b i a units corresponding to that of the overall promoter system.
- the weak promoter system comprises a lambda P L in combination with a single perfect palindrome operator sequence, most preferably a perfect palindrome lac operator, which is located downstream of the promoter and which overlaps the transcriptional start point.
- the operator has the sequence G G AATTGTG AG C GCTC ACAATTC C (SEQ ID NO 1) with the third nucleotide (highlighted in bold text) also representing the transcriptional start point.
- secretion leaders for periplasmic expression are well known in the art.
- the secretion leaders is chosen to secrete via the sec pathway.
- the secretion leader is chosen to secrete via the srp pathway.
- Examples of secretion leaders which can be employed include DsbA, ompA, pelB, and leaders having the sequences:
- a functionally equivalent sequence is one that shares 70% or greater identity with an amino acid sequence, preferably 75% or greater identity, more preferably 80% or greater identity and most preferably 90% or greater identity, such as 95% identity or more, and which retains the ability to secrete the target polypeptide from a prokaryotic cell.
- preferred secretion leaders are those having the sequences SEQ ID NO 2, SEQ ID NO 4 and especially SEQ ID NO 5.
- DNA sequences which are operably linked are contiguous and, in the case of a secretion leader, contiguous and in the same reading frame.
- the linkage between the secretion leader sequence and the CR 197 is such that the secretion leader sequence is attached to the N-terminal of the CRM197.
- the CRM197 is expressed with an N-terminal tag, in which case the linkage between the secretion leader sequence and the CRM 197 is such that the secretion leader sequence is attached to the tag, preferably to the N-terminus of the tag.
- the secretion leader sequence is preferably attached at the 5' end of the CR 197. Nucleotides encoding the sequence leaders are preferably codon optimised for expression in E coli.
- promoter and one or more perfect palindromic operators give rise to an inducible expression system, where expression of the CRM 197 is repressed or prevented until conditions are adjusted to induce expression.
- suitable induction conditions are well known in the art for the promoters and operators employed.
- One or more perfect palindrome operator sequences may be employed.
- Preferred operator sequences for use in the first and second aspects of the present invention are lac operators, and most preferably operators having the sequences GGAATTGTGAGCGCTCACAATTCC (SEQ ID NO 1) or AATTGTGAGCGCTCACAATT (SEQ ID NO. 7).
- SEQ ID NO. 7 When an operator of SEQ ID NO. 7 is employed, and overlaps the transcriptional start point, it is preferred that the first nucleotide (A) is the transcriptional start point.
- two perfect palindrome operator sequences are employed, most advantageously one operator sequence being located downstream of the promoter, and one operator sequence being located upstream of the promoter.
- the operator sequences are preferably spaced to maximise control of the promoter. In many embodiments, the spacing is from 85 to 150 base pairs apart, preferably from 90 to 126 base pairs apart, and most preferably 91 or 92 base pairs apart. In certain embodiments, an operator sequence overlaps with the transcriptional start point
- a single perfect palindrome operator sequence is employed, preferably downstream of the promoter, and most preferably the operator overlaps with the transcriptional start point
- Repressor sequences produce repressor protein, for example lacl gene sequence when using the lac operators.
- Other lac repressor sequences may also be used, for example the lacl Q sequence can be used to increase the level of (ac repressor protein.
- the repressor sequence may also be provided by the host cell genome or by using an additional compatible plasmid.
- the expression system may be integrated into the host cell genome, but is preferably comprised within an extrachromosomal element such as a plasmid.
- the expression system may be incorporated into phage or viral vectors and these used to deliver the expression system into the host cell system.
- Plasmids or expression vectors can be assembled by methods known in the art.
- the plasmid typically also comprises one or more of the following: a selectable marker, for example a sequence conferring antibiotic resistance, a cer stability sequence and an expression cassette.
- Expression may be induced by the addition of an inducer such as isopropyl-P-D-1- thiogalactopyranoside (IPTG), analogues of IPTG such as isobutyl-C-galactoside (IBCG), lactose or melibiose.
- inducers may be used and are described more fully elsewhere (e.g. see The Operon, eds Miller and Renznikoff (1978)). Inducers may be used individually or in combination. The construction of appropriate plasmids or expression vectors will be apparent to the scientist of ordinary skill.
- Preferred E coli strains are the B or K12 strains thereof.
- the expression system is expressed by methods well known in the art for the cells employed. Preferred expression methods include culturing the recombinant cells in growth medium, especially by fermentation, and then recovering the expressed protein.
- growth medium refers to a nutrient medium used for growing the recombinant cells. In many embodiments, a nutrient solution is employed. Suitable growth media for given recombinant cells are well known in the art.
- the host cells are cultured during a growth phase with conditions such that expression of the CRM197 is repressed or prevented.
- conditions are adjusted to induce expression of CRM197, for example by adding IPTG.
- conditions which have been found suitable for expression of CR 197 include growing the host cells at temperatures of from 30 to 40°C, preferably about 37°C.
- expression is induced at mid log phase.
- expression is induced with at a temperature of from 30 to 40°C, such as about 37°C.
- the temperature is reduced to a temperature less than 30°C, commonly no less than 20°C, such as about 25°C before expression is induced.
- expression of CRM197 is induced by addition of IPTG to give a concentration of up to 1mM IPTG, such as from 0.01 mM to 0.8mM IPTG, and especially about 0.5mM IPTG.
- expression of CRM197 is induced by addition of IPTG to give a concentration of up to 0.5mM IPTG, such as from 0.001 mM to 0.25mM IPTG and preferably about 0.01 mM IPTG.
- CRM197 can be purified by methods known in the art, see for example the processes described in WO2011/123139; US6,689,871 and Rappuoli et al, Journal of Chromatography, 268, 1983, pp 543 - 548.
- CR 197 is a non toxic mutant of Diphtheria toxin. See Nucleic Acids Res. 12 (10), 4063- 4069 (1984). The amino acid sequence was obtained from Genbank, accession number 10072 6A. All the following examples express this protein.
- CRM 197 A gene encoding CRM 197 was synthesised as an £.co//-optimised Ndel/Xhol fragment, and having the sequence:
- This fragment was cloned into plasmid pAVE011 , comprising a T7A3 promoter and two perfect-palindromic lac operators, prepared as described in international patent application WO 2007/088371 , using the Nde I and Xho I restriction sites in the vector. Recombinant clones were identified by restriction digest and confirmed by sequencing.
- One plasmid clone was transformed into E. coli W3110 strain CGSC4474 and the resulting strain designated CLD852. An equal amount of overnight culture was mixed with 40% glycerol and aliquoted into cryovials and stored at -70°C.
- This fragment was cloned into vector pAVE029, comprising a lambda P L promoter and a single palindromic operator downstream of the promoter, and overlapping the transcriptional start point, prepared as described in international patent application WO 2007/088371 , using the Nde I and Xho I restriction sites in the vector. Recombinant clones were identified by restriction digest and confirmed by sequencing. One plasmid clone was transformed into E. coli W3110 strain CGSC4474 and the resulting strain designated CLD846. An equal amount of overnight culture was mixed with 40% glycerol and aliquoted into cryovials and stored at -70°C.
- each flask was induced with IPTG (isopropyl-.p.-D-1-thiogalactopyranoside) to a final concentration 1.0mM, 0.1 mM or 0.01 mM and the incubation continued, under the conditions described above for 22 hours, during which samples were taken for measurement of growth, and accumulation of CRM197 within the bacterial cells.
- the accumulation levels of CRM197 was determined using SimplyBlue stained SDS-PAGE gels of whole cell lysates of the sampled.
- the harvested cells were further subjected to osmotic shock cell fractionation to isolate the cellular fraction containing proteins that had partitioned in the soluble E. coli periplasmic fraction.
- the OS1 (Osmotic Shock) fraction is the supernatant after washing in sucrose buffer, whilst the OS2 fraction is the supernatant after washing with a low ionic strength buffer. Whilst the OS2 is conventionally the released periplasmic proteins, it is often the case that the OS1 fraction also contains periplasmic protein.
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Abstract
In one aspect, a process for the intracellular expression of CRM197 in an E coli host is provided. The process comprises expressing a vector comprising a gene encoding CRM 197 operably linked to a promoter and at least one perfect palindrome operator sequence. In a related aspect, a process for the periplasmic expression of CRM197 in an E coli host is provided, comprising expressing a vector comprising a gene encoding CRM197 and a secretion leader sequence operably linked to a weak promoter system comprising at least one perfect palindrome operator sequence.
Description
PROCESS FOR EXPRESSION OF CRM197
The present invention concerns a process for the expression of recombinant CRM 197 in E. coli.
CRM 97 is a non-toxic mutant of diphtheria toxin having a single amino add substitution of glutamic acid for glycine. CRM197 is a well defined protein and functions as a carrier for polysaccharides and haptens making them immunogenic. It is utilized as a carrier protein in a number of approved conjugate vaccines for diseases such as meningitis and pneumococca) bacterial infections.
WO2011/123139 discloses the expression of CRM197 in Pseudomonus fluorescens, but reports that expression of this protein in E coli hosts is confined to impractically low amounts. Stefan et al, Journal of Biotechnology 156 (2011 ) pp245-252 discloses the expression of CRM197 in E coli using a PET9a vector. Expression was only achieved with a synthetic gene bearing an N-terminal his-tag. Expression without the tag always failed.
E. coli is a well characterised and commonly used host for the expression of recombinant proteins. Accordingly, it would be highly desirable to identify alternative methods of producing CRM 197 in E coli.
According to the first aspect of the present invention, there is provided a process for the intracellular expression of CRM197 in an E coli host, comprising expressing a vector comprising a gene encoding CRM197 operably (inked to a promoter and at least one perfect palindrome operator sequence.
Promoters which can be employed in the expression system of the present invention include phage RNA polymerase promoters and, commonly, E. coli RNA polymerase-based promoter systems. Examples of promoters which can be employed include T5, T7, T7A1 , T7A2, T7A3, λρΐ, pR, lac, lacUV5, trp, tac, trc, phoA and rrnB. Preferably, a strong promoter is employed in the first aspect of the present invention. Strong promoters include promoters having a strength in Pbia-units, measured according to the method given by Bujard et al, The EMBO Journal, Vol 5, No. 11 , pp2987-2994 (1986) of greater than about 10, and preferably greater than about 15, such as in the range of from 20 to 40 or greater. Preferred promoters include T7A , T7A2, T7A3 and T5.
Vectors and host cells comprising vectors for use in the process of first aspect also form part of the present invention.
According to a second aspect of the present invention, there is provided a process for the periplasmic expression of CRM 197 in an E coli host, comprising expressing a vector comprising a gene encoding CRM197 and a secretion leader sequence operably linked to a weak promoter system comprising at least one perfect palindrome operator sequence.
Weak promoter systems which can be employed in the second aspect of the present invention are those having a strength in Pbia-units, measured according to the method given by Bujard et al, The EMBO Journal, Vol 5, No. 11 , pp2987-2994 (1986) of
less than about 10, and preferably less than about 7, such as in the range of from 1 to 6. Examples of promoters which can be employed in the expression system of the second aspect of the present invention include phage RNA polymerase promoters and, commonly, E. coli RNA polymerase-based promoter systems. In certain embodiments, the promoters employed may be promoters which have a strength in Pbia units corresponding to that of the overall promoter system. Examples of promoters which can be employed include lac and lacUV5. In certain preferred embodiments, the weak promoter system comprises a lambda PL in combination with a single perfect palindrome operator sequence, most preferably a perfect palindrome lac operator, which is located downstream of the promoter and which overlaps the transcriptional start point. In many especially preferred embodiments, the operator has the sequence G G AATTGTG AG C GCTC ACAATTC C (SEQ ID NO 1) with the third nucleotide (highlighted in bold text) also representing the transcriptional start point.
Secretion leaders for periplasmic expression are well known in the art. In some embodiments, the secretion leaders is chosen to secrete via the sec pathway. In other embodiments, the secretion leader is chosen to secrete via the srp pathway. Examples of secretion leaders which can be employed include DsbA, ompA, pelB, and leaders having the sequences:
a) MLKRSSWLATLGLLTVASVSTIVYA (SEQ ID NO 2);
b) MKKATFITCLLAVLLVSNPIWNA (SEQ ID NO 3);
c) MKVSAAALAVILIATALCAPASA (SEQ ID NO 4);
d) MKVSTAFLCLLLTVSAFSAQVLA (SEQ ID NO 5); and
e) M CLLLALGLALACAAQA (SEQ ID NO 6)
or a functional equivalent thereof.
A functionally equivalent sequence is one that shares 70% or greater identity with an amino acid sequence, preferably 75% or greater identity, more preferably 80% or greater identity and most preferably 90% or greater identity, such as 95% identity or more, and which retains the ability to secrete the target polypeptide from a prokaryotic cell.
In certain embodiments of the second aspect of the present invention, preferred secretion leaders are those having the sequences SEQ ID NO 2, SEQ ID NO 4 and especially SEQ ID NO 5.
In many embodiments, DNA sequences which are operably linked are contiguous and, in the case of a secretion leader, contiguous and in the same reading frame.
Preferably, the linkage between the secretion leader sequence and the CR 197 is such that the secretion leader sequence is attached to the N-terminal of the CRM197. If desired, the CRM197 is expressed with an N-terminal tag, in which case the linkage between the secretion leader sequence and the CRM 197 is such that the secretion leader sequence is attached to the tag, preferably to the N-terminus of the tag.
The secretion leader sequence is preferably attached at the 5' end of the CR 197. Nucleotides encoding the sequence leaders are preferably codon optimised for expression in E coli.
The combination of promoter and one or more perfect palindromic operators give rise to an inducible expression system, where expression of the CRM 197 is repressed or prevented until conditions are adjusted to induce expression. Examples of suitable induction conditions are well known in the art for the promoters and operators employed.
Examples of operator sequences which may be employed in the expression system according to the present invention include lac, gal, deo and gin. One or more perfect palindrome operator sequences may be employed. Preferred operator sequences for use in the first and second aspects of the present invention are lac operators, and most preferably operators having the sequences GGAATTGTGAGCGCTCACAATTCC (SEQ ID NO 1) or AATTGTGAGCGCTCACAATT (SEQ ID NO. 7). When an operator of SEQ ID NO. 7 is employed, and overlaps the transcriptional start point, it is preferred that the first nucleotide (A) is the transcriptional start point.
In certain preferred embodiments of the first aspect of the present invention, two perfect palindrome operator sequences are employed, most advantageously one operator sequence being located downstream of the promoter, and one operator sequence being located upstream of the promoter. When two operator systems are employed, the operator sequences are preferably spaced to maximise control of the promoter. In many embodiments, the spacing is from 85 to 150 base pairs apart, preferably from 90 to 126 base pairs apart, and most preferably 91 or 92 base pairs apart. In certain embodiments, an operator sequence overlaps with the transcriptional start point
In certain preferred aspects of the second aspect of the present invention, a single perfect palindrome operator sequence is employed, preferably downstream of the promoter, and most preferably the operator overlaps with the transcriptional start point
It will be recognised that the operator system is commonly employed with an appropriate repressor sequence. Repressor sequences produce repressor protein, for example lacl gene sequence when using the lac operators. Other lac repressor sequences may also be used, for example the laclQ sequence can be used to increase the level of (ac repressor protein. The repressor sequence may also be provided by the host cell genome or by using an additional compatible plasmid.
The expression system may be integrated into the host cell genome, but is preferably comprised within an extrachromosomal element such as a plasmid. Alternatively, the expression system may be incorporated into phage or viral vectors and these used to deliver the expression system into the host cell system. Plasmids or expression vectors can be assembled by methods known in the art. The plasmid typically also comprises one or more of the following: a selectable marker, for example a
sequence conferring antibiotic resistance, a cer stability sequence and an expression cassette.
Expression may be induced by the addition of an inducer such as isopropyl-P-D-1- thiogalactopyranoside (IPTG), analogues of IPTG such as isobutyl-C-galactoside (IBCG), lactose or melibiose. Other inducers may be used and are described more fully elsewhere (e.g. see The Operon, eds Miller and Renznikoff (1978)). Inducers may be used individually or in combination. The construction of appropriate plasmids or expression vectors will be apparent to the scientist of ordinary skill.
Preferred E coli strains are the B or K12 strains thereof.
The expression system is expressed by methods well known in the art for the cells employed. Preferred expression methods include culturing the recombinant cells in growth medium, especially by fermentation, and then recovering the expressed protein. The term "growth medium" refers to a nutrient medium used for growing the recombinant cells. In many embodiments, a nutrient solution is employed. Suitable growth media for given recombinant cells are well known in the art.
In many preferred embodiments, the host cells are cultured during a growth phase with conditions such that expression of the CRM197 is repressed or prevented. When the host cells have reached a desired concentration, conditions are adjusted to induce expression of CRM197, for example by adding IPTG. Examples of conditions which have been found suitable for expression of CR 197 include growing the host cells at temperatures of from 30 to 40°C, preferably about 37°C. In many embodiments, expression is induced at mid log phase. In certain embodiments of the present invention, expression is induced with at a temperature of from 30 to 40°C, such as about 37°C. In certain embodiments of the second aspect of the present invention, the temperature is reduced to a temperature less than 30°C, commonly no less than 20°C, such as about 25°C before expression is induced.
In certain highly preferred embodiments of the first aspect of the present invention, expression of CRM197 is induced by addition of IPTG to give a concentration of up to 1mM IPTG, such as from 0.01 mM to 0.8mM IPTG, and especially about 0.5mM IPTG.
In many embodiments of the second aspect of invention, expression of CRM197 is induced by addition of IPTG to give a concentration of up to 0.5mM IPTG, such as from 0.001 mM to 0.25mM IPTG and preferably about 0.01 mM IPTG.
CRM197 can be purified by methods known in the art, see for example the processes described in WO2011/123139; US6,689,871 and Rappuoli et al, Journal of Chromatography, 268, 1983, pp 543 - 548.
The present invention is illustrated without limitation by the following examples.
Examples
CR 197 is a non toxic mutant of Diphtheria toxin. See Nucleic Acids Res. 12 (10), 4063- 4069 (1984). The amino acid sequence was obtained from Genbank, accession number 10072 6A. All the following examples express this protein.
Construction of strains CLD852
A gene encoding CRM 197 was synthesised as an £.co//-optimised Ndel/Xhol fragment, and having the sequence:
AGGAGGTAAAACATATGGGCGCAGACGACGTAGTAGACAGCAGCAAGAGCTTCGTG ATGGAAAATTTCAGCAGCTATCACGGTACCAAGCCGGGCTACGTTGACAGCATTCAA AAAGGCATCCAGAAGCCGAAAAGCGGCACCCAGGGTAATTACGACGATGACTGGAA AGAGTTCTATAGCACTGACAACAAGTACGACGCTGCTGGTTATTCCGTTGATAACGA GAACCCGCTGAGCGGTAAAGCGGGTGGTGTGGTCAAGGTTACGTATCCGGGCCTGA CCAAGGTCCTGGCGTTGAAGGTGGACAATGCGGAAACCATCAAGAAAGAGCTGGGC CTGTCCCTGACCGAGCCGCTGATGGAGCAAGTGGGTACGGAAGAGTTCATTAAACG TTTCGGTGATGGTGCATCGCGTGTGGTCTTGTCCTTGCCGTTCGCAGAAGGTAGCTC CAGCGTTGAGTACATCAATAACTGGGAACAGGCTAAGGCGCTGAGCGTCGAGCTGG AAATCAACTTTGAAACCCGTGGTAAACGTGGTCAAGATGCAATGTATGAGTACATGG CCCAGGCCTGCGCGGGTAATCGCGTCCGTCGCAGCGTTGGTTCTAGCCTGTCGTGC ATTAACCTGGATTGGGATGTCATTCGCGACAAGACGAAAACGAAAATTGAGTCTCTG AAAGAACATGGCCCGATTAAGAACAAAATGAGCGAGAGCCCGAACAAAACTGTTAGC GAAGAGAAGGCGAAGCAGTATCTGGAAGAATTTCACCAGACGGCTCTGGAACATCC GGAGCTGAGCGAGCTGAAAACGGTGACTGGCACGAATCCGGTGTTCGCCGGTGCC AACTATGCCGCGTGGGCGGTCAATGTGGCGCAGGTTATCGACAGCGAAACGGCGGA TAACCTGGAGAAAACCACCGCCGCATTGAGCATTCTGCCGGGCATTGGTAGCGTCAT GGGTATCGCGGATGGCGCCGTTCACCATAATACCGAAGAGATCGTCGCACAAAGCA TTGCACTGTCCAGCCTGATGGTGGCACAGGCAATCCCGCTGGTCGGTGAGCTGGTT GATATCGGTTTTGCTGCGTACAATTTTGTTGAAAGCATCATTAATCTGTTCCAGGTGG TTCACAATTCTTACAACCGTCCTGCGTACTCGCCGGGTCACAAAACCCAACCGTTCC TGCATGACGGCTACGCGGTTAGCTGGAATACCGTTGAAGATAGCATTATTCGCACCG GCTTCCAAGGCGAAAGCGGCCATGATATCAAAATCACGGCGGAGAATACCCCGCTG CCAATTGCAGGCGTCTTGCTGCCTACCATCCCGGGCAAGTTGGATGTGAACAAGAG CAAGACCCACATCAGCGTAAATGGTCGTAAAATCCGCATGCGCTGTCGTGCGATTGA CGGCGACGTGACGTTTTGTCGTCCGAAAAGCCCAGTGTATGTTGGTAACGGTGTGC ACGCGAATCTGCACGTTGCATTTCACCGTAGCAGCTCTGAGAAGATTCACTCCAACG AGATCAGCAGCGACAGCATTGGTGTCCTGGGTTACCAAAAGACCGTGGACCATACC
AAGGTTAACAGCAAACTGAGCCTGTTTTTCGAGATCAAGTCTTAACTCGAG (SEQ ID NO 8)
This fragment was cloned into plasmid pAVE011 , comprising a T7A3 promoter and two perfect-palindromic lac operators, prepared as described in international patent application WO 2007/088371 , using the Nde I and Xho I restriction sites in the vector. Recombinant clones were identified by restriction digest and confirmed by sequencing. One plasmid clone was transformed into E. coli W3110 strain CGSC4474 and the resulting strain designated CLD852. An equal amount of overnight culture was mixed with 40% glycerol and aliquoted into cryovials and stored at -70°C.
CLD846
A gene encoding CRM197 together with an N-termina) secretion leader of Seq ID No.4 was synthesised as an Eco//-optimised Ndel/Xhol fragment having the sequence:
AGGAGGTAAAACATATGAAGGTATCTACTGCGTTTCTCTGTCTGTTGCTCACTGTATC TGCATTTTCGGCTCAGGTCTTGGCTGGCGCAGACGACGTAGTAGACAGCAGCAAGA GCTTCGTGATGGAAAATTTCAGCAGCTATCACGGTACCAAGCCGGGCTACGTTGACA GCATTCAAAAAGGCATCCAGAAGCCGAAAAGCGGCACCCAGGGTAATTACGACGAT GACTGGAAAGAGTTCTATAGCACTGACAACAAGTACGACGCTGCTGGTTATTCCGTT GATAACGAGAACCCGCTGAGCGGTAAAGCGGGTGGTGTGGTCAAGGTTACGTATCC GGGCCTGACCAAGGTCCTGGCGTTGAAGGTGGACAATGCGGAAACCATCAAGAAAG AGCTGGGCCTGTCCCTGACCGAGCCGCTGATGGAGCAAGTGGGTACGGAAGAGTTC ATTAAACGTTTCGGTGATGGTGCATCGCGTGTGGTCTTGTCCTTGCCGTTCGCAGAA GGTAGCTCCAGCGTTGAGTACATCAATAACTGGGAACAGGCTAAGGCGCTGAGCGT CGAGCTGGAAATCAACTTTGAAACCCGTGGTAAACGTGGTCAAGATGCAATGTATGA GTACATGGCCCAGGCCTGCGCGGGTAATCGCGTCCGTCGCAGCGTTGGTTCTAGCC TGTCGTGCATTAACCTGGATTGGGATGTCATTCGCGACAAGACGAAAACGAAAATTG AGTCTCTGAAAGAACATGGCCCGATTAAGAACAAAATGAGCGAGAGCCCGAACAAAA CTGTTAGCGAAGAGAAGGCGAAGCAGTATCTGGAAGAATTTCACCAGACGGCTCTG GAACATCCGGAGCTGAGCGAGCTGAAAACGGTGACTGGCACGAATCCGGTGTTCGC CGGTGCCAACTATGCCGCGTGGGCGGTCAATGTGGCGCAGGTTATCGACAGCGAAA CGGCGGATAACCTGGAGAAAACCACCGCCGCATTGAGCATTCTGCCGGGCATTGGT AGCGTCATGGGTATCGCGGATGGCGCCGTTCACCATAATACCGAAGAGATCGTCGC ACAAAGCATTGCACTGTCCAGCCTGATGGTGGCACAGGCAATCCCGCTGGTCGGTG AGCTGGTTGATATCGGTTTTGCTGCGTACAATTTTGTTGAAAGCATCATTAATCTGTT CCAGGTGGTTCACAATTCTTACAACCGTCCTGCGTACTCGCCGGGTCACAAAACCCA ACCGTTCCTGCATGACGGCTACGCGGTTAGCTGGAATACCGTTGAAGATAGCATTAT TCGCACCGGCTTCCAAGGCGAAAGCGGCCATGATATCAAAATCACGGCGGAGAATA
CCCCGCTGCCAATTGCAGGCGTCTTGCTGCCTACCATCCCGGGCAAGTTGGATGTG AACAAGAGCAAGACCCACATCAGCGTAAATGGTCGTAAAATCCGCATGCGCTGTCGT GCGATTGACGGCGACGTGACGTTTTGTCGTCCGAAAAGCCCAGTGTATGTTGGTAAC GGTGTGCACGCGAATCTGCACGTTGCATTTCACCGTAGCAGCTCTGAGAAGATTCAC TCCAACGAGATCAGCAGCGACAGCATTGGTGTCCTGGGTTACCAAAAGACCGTGGA CCATACCAAGGTTAACAGCAAACTGAGCCTGTTTTTCGAGATCAAGTCTTAACTCGAG
(SEQ ID NO 9)
This fragment was cloned into vector pAVE029, comprising a lambda PL promoter and a single palindromic operator downstream of the promoter, and overlapping the transcriptional start point, prepared as described in international patent application WO 2007/088371 , using the Nde I and Xho I restriction sites in the vector. Recombinant clones were identified by restriction digest and confirmed by sequencing. One plasmid clone was transformed into E. coli W3110 strain CGSC4474 and the resulting strain designated CLD846. An equal amount of overnight culture was mixed with 40% glycerol and aliquoted into cryovials and stored at -70°C.
Example 1. Shakeflask evaluation of CLD852 - Induction at 37°C
10μΙ of the thawed glycerol stock of CLD852 was inoculated into 5ml Luria Broth (LB, 5g/L yeast extract, 10g/L tryptone, and 5g/L sodium chloride) supplemented with tetracycline (10pg/ml). This was incubated at 37°C in an orbital shaker for 16h. 500μΙ of this culture was then used to inoculate one 250ml Erlenmeyer flasks containing 50ml of Luria Broth (composition as described above). The flasks were incubated at 37°C, at 200rpm in an orbital shaker. Growth was monitored until OD6Q0=0.5-0.7. At this point the flask was induced with IPTG (isopropyl-.p.-D-l-thiogalactopyranoside) to a final concentration lOm and the incubation continued, under the conditions described above for 22 hours, during which samples were taken for measurement of growth, and accumulation of CR 197 within the bacterial cells. The accumulation levels of CR 197 was determined using SimplyBlue stained SDS-PAGE gels of whole cell lysates of the sampled. The harvested cells were further subjected to Bugbuster cell disruption to analyse whether the CRM197 was soluble or insoluble.
Analysis of the total culture by SDS-PAGE showed that substantial amounts of a protein of the expected molecular weight for CRM197 was made. The protein of the expected molecular weight reacted to the Anti-diphtheria/CRM197 antibody on Western blot confirming CR 197 was made. Solubility analysis showed that this material was found exclusively in the form of inclusion bodies.
Example 2. Shakeflask evaluation of CLD852 - Induction at 25°C
10μΙ of the thawed glycerol stock of CLD852 was inoculated into 5ml Luria Broth (LB, 5g/L yeast extract, 10g/L tryptone, and 5g/L sodium chloride) supplemented with tetracycline (10pg/ml). This was incubated at 37°C in an orbital shaker for 16h. 500μΙ of this culture was then used to inoculate two 250ml Erlenmeyer flasks containing 50ml of Luria Broth (composition as described above). The flasks were incubated at 37°C, at 200rpm in an orbital shaker. Growth was monitored until OD600=0.5-0.7. At this point the temperature was lowered to 25°C and the flasks were induced with IPTG (isopropyl-. .-D- 1-thiogalactopyranoside) to a final concentration of 0.5mM or 0.1 m and the incubation continued, under the conditions described above for 22 hours, during which samples were taken for measurement of growth, and accumulation of CRM 197 within the bacterial cells. The accumulation levels of CRM197 was determined using SimplyBlue stained SDS- PAGE gels of whole cell lysates of the sampled. The harvested cells were further subjected to Bugbuster cell disruption to analyse whether the CRM 97 was soluble or insoluble.
Analysis of the total culture by SDS-PAGE showed that substantial amounts of a protein of the expected molecular weight for CRM 197 was made. The protein of the expected molecular weight reacted to the Anti-diphtheria/CRM 197 antibody on Western blot confirming CRM197 was made. Surprisingly, solubility analysis showed that this material was found equally as soluble material and as inclusion bodies.
Example 3. Shake-flask evaluation of CLD846
10μΙ of the thawed glycerol stock of CLD846 was inoculated into 5ml Luria Broth (LB, 5g/L yeast extract, 10g/L tryptone, and 5g/L sodium chloride) supplemented with tetracycline (10pg/ml). This was incubated at 37°C in an orbital shaker for 16h. 500μΙ of this culture was then used to inoculate three 250ml Erlenmeyer flasks containing 50ml of Luria Broth (composition as described above). The flasks were incubated at 37°C, at 200rpm in an orbital shaker. Growth was monitored until OD600 = 0.5 - 0.7. At this point each flask was induced with IPTG (isopropyl-.p.-D-1-thiogalactopyranoside) to a final concentration 1.0mM, 0.1 mM or 0.01 mM and the incubation continued, under the conditions described above for 22 hours, during which samples were taken for measurement of growth, and accumulation of CRM197 within the bacterial cells. The accumulation levels of CRM197 was determined using SimplyBlue stained SDS-PAGE gels of whole cell lysates of the sampled. The harvested cells were further subjected to osmotic shock cell fractionation to isolate the cellular fraction containing proteins that had partitioned in the soluble E. coli periplasmic fraction. The OS1 (Osmotic Shock) fraction is the supernatant after washing in sucrose buffer, whilst the OS2 fraction is the supernatant after washing with a low ionic
strength buffer. Whilst the OS2 is conventionally the released periplasmic proteins, it is often the case that the OS1 fraction also contains periplasmic protein.
Analysis of the total culture by SDS-PAGE showed that substantial amounts of a protein of the expected molecular weight for CRM 197 was made. The protein of the expected molecular weight reacted to the Anti-diphtheria/CRM197 antibody on Western blot confirming CR 197 was made. Location analysis shows that this material is found solely in the OS2 fraction.
Claims
1. A process for the intracellular expression of CRM 197 in an E coli host, comprising expressing a vector comprising a gene encoding CRM197 operably linked to a promoter and at least one perfect palindrome operator sequence.
2. A process according to claim 1 , wherein the promoter is an E. coli RNA polymerase-based promoter, and preferably a T7A3 promoter
3. A process according to either claim 1 or 2, wherein two perfect palindrome operator sequences are employed, preferably one operator sequence being located downstream of the promoter, and one operator sequence being located upstream of the promoter.
4. A process according to claim 3, wherein two operators are employed and the operator sequences are spaced 91 or 92 base pairs apart.
5. A process according to any preceding claim wherein an operator downstream of the promoter overlaps the transcriptional start point.
6. A process according to any preceding claim, wherein expression is induced at a temperature of from 30 to 40°C.
7. A process according to any one of claims 1 to 5, wherein expression is induced at a temperature of no less than 20°C and less than 30 °C.
8. A vector comprising a gene encoding CRM197 operably linked to a promoter and at least one perfect palindrome operator sequence.
9. An E. coli host cell transformed with a vector according to claim 8.
10. A process for the periplastic expression of CRM 197 in an £ coli host, comprising expressing a vector comprising a gene encoding CRM197 and a secretion leader sequence operably linked to a weak promoter system comprising at least one perfect palindrome operator sequence.
11. A process according to claim 10, wherein the weak promoter system comprises a lambda PL in combination with a single perfect palindrome operator sequence.
12. A process according to claim 10 or 1 1, wherein the operator is a perfect palindromic lac operator.
13. A process according to claim 12, wherein expression is induced by addition of IPTG to give a concentration of from 0.001 mM to 0.25mM IPTG and preferably about 0.01 mM IPTG.
14. A vector comprising expressing a vector comprising a gene encoding CRM197 and a secretion leader sequence operably linked to a weak promoter system comprising at least one perfect palindrome operator sequence.
15. An E. coli host cell transformed with a vector according to claim 14,
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WO2016079755A1 (en) | 2014-11-20 | 2016-05-26 | Biological E Limited | Codon optimized polynucleotide for high level expression of crm197 |
AU2015210674B2 (en) * | 2014-01-31 | 2017-08-24 | Fina Biosolutions, Llc | Expression and purification of CRM197 and related proteins |
WO2018172739A1 (en) * | 2017-03-24 | 2018-09-27 | Fujifilm Diosynth Biotechnologies Uk Limited | Expression system |
US11060123B2 (en) | 2014-01-31 | 2021-07-13 | Fina Biosolutions, Llc | Production of soluble recombinant protein without n-terminal methionine |
RU2803949C1 (en) * | 2019-09-03 | 2023-09-22 | Дженофокус Ко., Лтд. | Method for crm197 protein expression |
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AU2015210674B2 (en) * | 2014-01-31 | 2017-08-24 | Fina Biosolutions, Llc | Expression and purification of CRM197 and related proteins |
US10093704B2 (en) | 2014-01-31 | 2018-10-09 | Fina Biosolutions, Llc | Expression and purification of CRM197 and related proteins |
US11060123B2 (en) | 2014-01-31 | 2021-07-13 | Fina Biosolutions, Llc | Production of soluble recombinant protein without n-terminal methionine |
WO2016079755A1 (en) | 2014-11-20 | 2016-05-26 | Biological E Limited | Codon optimized polynucleotide for high level expression of crm197 |
US10280409B2 (en) | 2014-11-20 | 2019-05-07 | Biological E Limited | Codon optimized polynucleotide for high level expression of CRM197 |
WO2018172739A1 (en) * | 2017-03-24 | 2018-09-27 | Fujifilm Diosynth Biotechnologies Uk Limited | Expression system |
JP2020511151A (en) * | 2017-03-24 | 2020-04-16 | フジフィルム・ダイオシンス・バイオテクノロジーズ ・ユーケイ・リミテッド | Expression system |
US11479776B2 (en) | 2017-03-24 | 2022-10-25 | Fujifilm Diosynth Biotechnologies Uk Limited | Expression system |
JP7443061B2 (en) | 2017-03-24 | 2024-03-05 | フジフィルム・ダイオシンス・バイオテクノロジーズ ・ユーケイ・リミテッド | expression system |
RU2803949C1 (en) * | 2019-09-03 | 2023-09-22 | Дженофокус Ко., Лтд. | Method for crm197 protein expression |
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