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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • 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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

• This invention relates to modified host cells for expressing proteins and methods relating thereto by improved expression of tRNAs.
• the present invention is related to a host cell with one or more rRNA operons that comprise one or more polynucleotides encoding a tRNA.
• the polynucleotide encoding the tRNA may be derived from a human, yeast, or from the genome of the host cell.
• the polynucleotide encoding the tRNA may comprise any sequence of SEQ ID NOS: 1-7.
• the polynucleotide encoding the tRNA may comprise SEQ ID NOS: 1 and 7.
• the polynucleotide encoding the tRNA may comprise SEQ ID NOS: 1, 5 and 6.
• the polynucleotide encoding the tRNA may comprise SEQ ID NOS: 1, 5, 6 and 7.
• the polynucleotide encoding the tRNA may comprise SEQ ID NOS: 1-7.
• the host cell may be a bacteria, yeast or insect cell.
• the host cell may also be an E. coli.
• the rRNA operon of the host cell comprising the polynucleotide encoding the tRNA may be one or more of rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH.
• the polynucleotide encoding the tRNA may be '3 of 5S rRNA elements and 5' of terminator elements.
• the host cell may also comprise a gene encoding a heterologous protein.
• the heterologous gene may comprise one or more codons with rare tRNAs in the precursor of the host cell.
• the present invention is also related to a method of producing a recombinant heterologous protein cell.
• a gene encoding the heterologous protein is expressed in the host cell described herein.
• Figure 1 shows the codon frequency of E. coli and M. tuberculosis, as well as the tRNA frequency of E. coli.
• Figure 2 depicts a tRNA cloverleaf secondary structure with the base positions labeled in the standard numbering system.
• the bases represented by ovals are variable in that they may or may not be present.
• the present invention provides methods and compositions for increased expression of proteins in a host cell, characterized by a host cell comprising a tRNA gene in the rRNA operon of the host cell.
• the rRNA operon is a desirable destination for the introduction of a tRNA gene, because rRNA operons have strong promoters. This is evidenced by rRNA being the major component of cellular RNA.
• polynucleotides may be added to an rRNA operon without interfering with expression and processing of rRNAs.
• the present invention is related to the introduction of one or more tRNA genes into the chromosome of a host cell.
• Each tRNA gene may be introduced into one or more locations of one or more rRNA operons of the host cell.
• the tRNA gene introduced into the rRNA operon of the host cell may be chosen based on the frequency of the codons present in the gene encoding the protein of interest compared with the frequency of codons or the frequency of cognate tRNAs expressed in the host cell.
• a rare tRNA gene encoding a rare tRNA or encoding a cognate tRNA of a rare codon may be introduced into an rRNA operon of the host cell.
• genes for one or more of such cognate tRNAs may be inserted into the rRNA operon of the host cell. All tRNAs or any portion thereof, without regard to their prevalence, may also introduced into the rRNA operon, which may increase tRNA levels for all tRNAs.
• a rare codon may be any codon in the host cell that is present in the host cell at a frequency less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 per thousand.
• a rare tRNA may be any tRNA with cell amounts, relative to Leu-tRNA, less than 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.1, 0.09. 0.08. 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01.
• Figure 1 lists the codon frequency of E. coli and M. tuberculosis, as well as the tRNA frequency of E. coli.
• the rare tRNA gene may be derived from the tRNA gene of the host cell.
• the native rare tRNA may be cloned or synthesized and inserted into the rRNA operon of the host cell using methods known in the art.
• the rare tRNA gene may also be derived from another organism, such as the source of the gene encoding the protein of interest.
• the rare tRNA may also be a synthetic, mutated, modified, suppressor, viral or bacteriophage tRNA. [0019]
• the rare tRNA may be modified in the non-constant portions of the tRNA.
• the non-constant portions may be modified by substitutions or deletions.
• Figure 1 depicts the secondary structure of a tRNA with those positions that may be deleted indicated by ovals (17, 17 A, 2OA and 2OB in the D Loop and the bases between 45 and 46 in the Variable Region).
• the rare tRNA may also be a tRNA for introducing an unnatural amino acid, as described in U.S. Patent Publication No. 20040265952.
• tRNA genes that may be used include the E. coli genes listed in Table 1. Table 1
• Each rRNA operon is a polycistronic suite of genes.
• An rRNA operon may comprise combinations of DNA encoding ribosomal RNAs (16S rRNA, 23 S rRNA, and 5 S rRNA), spacer tRNAs, distal tRNAs, and regulatory elements.
• a schematic illustration of the seven polycistronic rRNA operons of E. coli is presented in Table 2.
• the one or more rare tRNA genes may be introduced individually or together anywhere in the operon that allows for their expression.
• the rare tRNA genes may be introduced into the rRNA operon 3 ' of Pi or P 2 .
• the rare tRNA genes may be introduced between the components of the rRNA operon (intergenic) or within a components of the rRNA operon (intragenic).
• the operon and the position on the operon at which to insert the rare tRNA genes one of skill in the art may consider the likelihood of homologous recombination events.
• the site of introduction of a tRNA may result in homologous recombination between rRNA operons, due to the high degree of similarity between rRNA operons.
• the rare tRNAs may be inserted in the 5 S distal regions of the rRNA operon.
• the rrn operons of E. coli show considerable variation in their 5 S distal regions, located at the 3' end of each operon.
• a genetic marker has been successfully introduced as a replacement for rrfli without negative effect on expression on the rrnH operon. (Ammons, et al. 1998).
• the rrnA, rrnB, rrnE, and rrnG operons are also candidates for the addition of rare tRNA genes since they do not already include distal tRNAs. 3.
• the present invention is also related to a host cell comprising one or more rare tRNA genes in one or more rRNA operons of the host cell.
• the amount of rare tRNA present in the host cell is increased compared to the levels of rare tRNA in the parent cell.
• the amount of rare tRNA in the host cell may be increased by greater than 1.5-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5- or 5.0-fold.
• the host cell may be derived from any parent cell which is suitable for expression of the protein of interest.
• the parent cell may be either eukaryotic or prokaryotic, which may be Gram- positive or Gram-negative.
• Representative examples of parent cells include, but are not limited to, yeast, E. coli, B. subtilis, Streptomyces, Proteus mirabilis, Staphylococcus, such as e.g. S 1 . carnosus, and other cells available from public collections.
• the parent cell may be a bacteria, which may be a reduced genome bacterium as described in U.S. Patent Application No. 10/057,582 and in Blattner, (2004), which are both incorporated by reference herein. 4. Method of Making Host Cell
• the present invention is also related to a method of making a host cell.
• a host cell may be made by introducing one or more rare tRNA genes into one or more rRNA operons of the parent cell.
• a tRNA gene may be introduced into an rRNA operon by methods well known in the art, such as may be found in Sambrook et al. , "Molecular Cloning Manual, " Cold Spring Harbor Laboratory (2001).
• the present invention is also related to an expressible host cell comprising a host cell of the present invention which comprises a heterologous gene encoding a protein of interest.
• the heterologous gene may be operatively linked to a promoter element.
• the heterologous gene may be located within the chromosome of the expressible host cell.
• the heterologous gene may be inserted into the chromosome of the expressible host cell using methods well known in the art, such as those methods described in Sambrook et al. (2001).
• the heterologous gene may also be located on a vector, with the expressible host cell comprising the vector.
• the vector may be capable of autonomous replication or it may integrate into the DNA of the microorganism.
• the vector may be any polynucleotide that is capable of transporting the heterologous gene including, but not limited to, a plasmid, cosmid, phage, phagemid, YAC or viral vector.
• the vector may be in a form suitable for expression of the heterologous gene in the expressible host cell.
• the expression vectors may be designed for expression of the protein of interest in the host cell.
• the protein of interest may be any protein known in the art, such as, for example, insulin, hGH, tPA, cytokines, such as e.g. interleukins (IL) such as IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, Ih-I, IL-8, IL-9, IL-10, IL-I l, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumour necrosis factor (TNF), TNF alpha and TNF beta, TRAIL, G-CSF, GM-CSF, M-CSF, MCP-I, VEGF and antibodies.
• IL interleukins
• IFN interferon alpha, IFN beta, IFN gamma, IFN omega or IFN tau
• TNF tumour necrosis factor
• the present invention is also related to a method of producing a protein of interest comprising isolating the protein of interest from an expressible host cell comprising a polynucleotide encoding the protein of interest. Methods for optimizing protein expression are discussed in Hannig et al, Trends Biotechnol., 16(2):54-60 (1998), the contents of which are hereby incorporated by reference in their entirety.
• the expressible host cell may constitutively express the polynucleotide encoding the protein of interest.
• Expression of the polynucleotide may also be regulated by one or more operatively linked regulatory sequences including, but not limited to, promoters, enhancers and other expression control elements (e.g., polyadenylation signals).
• Inducible regulatory sequences may be activated or repressed based on the local environment of the expressible host cell. 7. Kits
• the present invention is also related to a kit comprising a container comprising host cells of the present invention.
• the host cells may be competent cells.
• the kit may optionally comprise one or more plasmids, one or more reagents for use in transformation, or instructions for use of the host cells in producing expressible host cells or proteins of interest.
• the present invention is also related to a kit comprising a container comprising expressible host cells of the present invention.
• the expressible host cells may comprise a polynucleotide encoding a protein of interest.
• the kit may optionally instructions for propagating the cells or using the cells for producing the protein of interest.
• the present invention has multiple aspects, illustrated by the following non-limiting example.
• DHPS P. falciparum dihydropteroate synthetase
• E. coli host cells of strain MDS42 are transformed by inserting argU and UeX genes into the chromosome at the 3' end of the rrnG operon.
• the resulting tRNA-augmented host cells are also transformed with an expression vector comprising P. falciparum dihydropteroate synthetase (DHPS), located downstream of a promoter, along with an antibiotic resistance marker for ampicillin.
• DHPS P. falciparum dihydropteroate synthetase
• a second sample of unmodified MDS42 E. coli cells are transformed with the same expression vector as a control.
• Overnight cultures are prepared by picking single colonies of transformed tRNA- augmented host cells and inoculating 3 ml Luria-Bertani (LB) media supplemented with ampicillin (100 ⁇ g/ml). The following morning, 100 ⁇ l of the overnight culture are used to inoculate flasks containing 2 ml of LB supplemented with ampicillin (100 ⁇ g/ml). These cultures are incubated for 3-4 hours at 37 0 C until cell density OD 600 reaches 0.5-0.7. The cultures are induced by the addition of IPTG to a final concentration of 0.5mM and then harvested 3 hours after induction. Expression of protein is monitored by SDS-PAGE of pre- induction and harvest whole cell lysates of cells with and without tRNA augmentation. Western analysis is performed on P. falciparum DHPS to confirm protein identity.
• LB Luria-Bertani
• Enzyme assays on the tRNA-augmented cells compared with the non-augmented control cells indicate that expression of P. falciparum DHPS is greatly enhanced in the tRNA augmented cells.

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• 2007
  • 2007-04-23 AT AT07761135T patent/ATE473278T1/de not_active IP Right Cessation
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