Submitted to the School of Nano-Bioscience and Chemical Engineering and the Graduate School of UNIST in fulfillment of the degree of. Various plant-derived beneficial chemicals have been used for humans from ancient times to modern times. Due to differences in genetic characters between bacteria and plant, it may cause low plant protein expression or formation of inclusion bodies composed of insoluble protein precipitates.
To alter rare tRNA abundance and chaperone expression level, we performed chromosomally integrated promoter mutant libraries using recombination with single-stranded DNA oligonucleotides. The tRNA and chaperone promoter mutants also showed significant increases in target Arabidopsis protein expression.
Introduction
- Escherichia coli as an industrial bacterial host for heterologous protein expression
- Difference of codon usage bias between E. coli and Arabidopsis thaliana
- Prevention of inclusion body formation by molecular chaperones
- Simultaneous genome editing for introduction of various mutation
- Optimal expression of plant genes in E. coli
Codons are recognized by anticodon of complementary tRNAs loaded with the amino acid.21 The degeneracy of the genetic code allows many alternative DNA sequences to encode the same protein sequence.22 Codon distribution is related to genome GC content and the changes in codon usage bias can be at least partially explained by a mutation-selection equilibrium between the different synonymous codons in each organism.9 According to arithmetic result of genome analysis of E. A complete summary of codon usages can be found at http://www. kazusa.or.jp/codon/. Chaperones are one of the systems for synthesis and maintenance of protein in abnormal state.32 The main function of chaperones is to prevent both synthesized polypeptide chains and assembled subunits from assembling into non-functional structures.32 Most chaperones are expressed in heat-shocked conditions because chaperones has specific promoters corresponding to sigma factor 32 (σ32) which is expressed by heat shock, not sigma factor 70 (σ70) which is expressed constitutively.33 Because of this region, expression of chaperones is low in normal condition.33 In heat shock condition, cytosolic and membrane proteins are damaged and aggregated in the cell and this can cause cell death.34 Chaperones help new protein synthesis and damaged protein repair.35 In particular, DnaKJ and GrpE system help polypeptide elongation in protein synthesis, and GroELS system helps transfer misfolded protein to functional protein to switch by refolding.36, 37.
The simultaneous engineering of multiple sites in the genome using short single-stranded oligonucleotides, called multiplex automated genome engineering (MAGE), has currently become a unique genome engineering tool that could help in efficient and accelerated mutagenesis of desired functions in a short time.40 -42 This technique uses the lambda RED (Gamma, Exo, Beta) recombination system and short nucleotide oligos to introduce the desired mutations to E. 40-42 Inactivation of the methyl mismatch repair (MMR) system is also required. avoid correction of engineered regions with the native MMR system.43 Although several modified E. If the effect of different codon usage deviations in heterologous protein expression manifests as a negative result with different tRNA levels, one solution is to expand the host intracellular tRNA pool.9 For E.
First, the expression of rare tRNA extra copies is associated with adjusting the codon usage bias between E.
Materials and methods
- Bacterial strains and growth conditions
- Strain construction
- Plasmid construction
- Multiplex automated genome engineering (MAGE)
- High-throughput screening (HTS) for desired mutants after MAGE
- SDS-PAGE and western blotting of target proteins
- Heterologous expression of other plant genes in engineered E. coli strains
Cells were cooled immediately after induction to arrest the metabolic state of the cell, made electrocompetent, and transformed with the PCR products as described above. SOC medium was added after electroporation, and cells were electroporated by incubation at 30°C for 2 h. All DNA manipulations were performed using established protocols.63 To confirm the state of Arabidopsis protein expression in E .
Primers were designed by inserting the restriction enzyme site corresponding to the pET30a(+) restriction enzyme site. Reverse primers include XhoI (CTCGAG, NEB biolabs), NotI (GCGGCCGC, NEB biolabs), HindIII (AAGCTT, NEB biolabs) restriction enzymes. To confirm the insertion of the desired gene into the pET30a(+) vector, colony PCR and sequencing (Marcrogen Inc.) were performed.
To confirm Arabidopsis protein expression in MAGE mutant cells, pBbB6a (Biobrick plasmid backbone) was used as the expression vector. Because the BamHI restriction enzyme site is located in HIS6, B6a-HIS6_RP including the XhoI restriction enzyme site was used as the reverse primer. After cooling, about 1 ml ~ 2 ml of cells were centrifuged in a pre-cooled centrifuge at 4°C, and then the supernatant was removed.
The transformed cells were obtained in 3 ml of modified LB supplemented with appropriate chloramphenicol and kanamycin in a test tube until the OD600 reached 0.5~0.6 at 30 °C. In order to obtain cells containing the desired MAGE mutation, a screening method must be established because the desired mutants in the mutant mixture are small.40 After MAGE recovery, the seed was inoculated using a 1/100 dilution in M9 minimal medium (0.2% glucose) supplemented with the appropriate chloramphenicol and kanamycin to enrich for mutant MAGE cells. The mutant mixture, diluted to 10 3 cells, was plated on an M9 plate to separate individual mutants.
Single mutant cells were inoculated into 300 µl LB medium with appropriate antibiotics in 2 ml 96-well microplates, respectively. Based on TECAN growth data, high-growth cells were arranged and 4 high-growth strains were grown in 30 ml of M9 medium in culture flasks. To compare the amplification of engineered strains, wild type with pBbB6a-gDS was made and the protein expression of gDS was analyzed by SDS-PAGE and Western blotting.
Results
- Replacement of E. coli native genes with their corresponding A. thaliana gene
- Genome engineering of rare tRNA promoters to enhance tRNA expression
- Genome engineering of chaperone promoters
- Simultaneous genome engineering of rare tRNA and chaperone promoters
- Protein expression level of engineered strains
- Versatility of effective plant gene expression strains
Because essential enzymes for the biosynthesis of amino acids on the genome were removed, protein synthesis for cell growth was suppressed. However, if there are problems with Arabidopsis protein expression, replacement strains may not grow well in M9 media. As shown in the figure, the replacement strains of TSA1, ADT2 and HIS6 showed low growth in M9 media, together with knockout strains.
If TSA1, ADT2, and HIS6 proteins are well expressed, we expect replacement strains to grow well in M9 media. In this case, these mutations could aid in the soluble and functional expression of the Arabidopsis protein in E. In Figure 7A, the TSA1 and ADT2 tRNA MAGE mutant mixture showed a slight increase in growth in M9 minimal media. individual mutants of TSA1-tRNA MAGE mutants showed a slight increase in growth.
Individual mutants were sequenced by TECAN. left) 4 high-ranked strains were analyzed by OD600. right). When individual mutants were separated, many mutants showed higher growth than no-MAGE TSA1 strain. Using this rapidly growing TSA1 chaperone MAGE mutant, TSA1 protein overexpression was performed by IPTG induction of pBbB6a-TSA1.
To get comprehensive improvement of Arabidopsis protein expression, promoter changes of rare tRNA genes (such as argU, ileY, leuW, ileX and argW) and chaperone genes (such as dnaK-dnaJ, grpE, groEL-groES) were performed by co. - STOMACH. In MAGE mutant mixture, TSA1 tRNA+chaperone MAGE showed increase of growth and HIS6 tRNA+chaperone MAGE showed increase of growth compared to no-MAGE HIS6 strain. After MAGE, some mutants showed increase in growth compared to no-MAGE strain in M9 minimal media.
Confirmation of protein expression of MG-MAGE strains harboring the Arabidopsis gene was performed for comparison between wild-type and engineered strains. In the results of Figure 10B, TSA1 protein expression of TcM2 and TcM3 strains was shown as soluble expression.
Discussion
Third, the concentration of rare tRNAs and chaperones is not sufficient to address differences in codon usage bias and misfolded protein refolding. Although these promoter mutations are constitutive promoters, there is no guarantee of the ability to necessary tRNA and chaperone concentration. This can be overcome by adding extra copies of tRNA and chaperone on the genome or adding additional strong promoter near the original promoter.
Until now, many bacterial hosts were optimized for heterologous protein production.8 Primarily, all bacteria could be used for heterologous protein production.8 Many genomic studies provide new information about the bacterial hosts that are frequently or rarely used.65 Infrequently used codons can be a reason to change from an E. Heterologous protein expression involves the analysis of genes and the transfer of the corresponding DNA fragments into hosts other than the original DNA fragments encoding proteins.10 Protein isolation, directly from plant proteins, can be expensive, difficult to process and take a long time, and heterologous expression provides a convenient way.66 This method allows large-scale production of plant-derived proteins in E.
Conclusions
Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Recombinant protein folding and misfolding in Escherichia coli. enhanced by harmonizing the codon usage frequencies of the target gene with that of the expression host. Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the significant species diversity.
Protein evolution and gene expression levels are associated in Drosophila and are independently associated with mRNA abundance, protein length, and number of protein-protein interactions. Advances in the refolding of proteins produced in E. Biology of heat shock proteins and molecular chaperones. The function of heat shock proteins in stress tolerance: degradation and reactivation of damaged proteins.
Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Codon-defined ribosomal pausing in Escherichia coli revealed using the pyRE attenuator to probe the link between transcription and translation. Silent mutations in the leader peptide region of Escherichia coli ompA strongly affect transcription and translation in vivo.
Heterologous protein production in Escherichia coli: an investigation into the effect of codon usage and optimization of the expression host. Chaperone co-expression plasmids: Differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of a Japanese cedar pollen allergen, Cryj2, in Escherichia coli. Protein overproduction in< i> Escherichia coli: Mutant hosts that allow synthesis of several membrane proteins and globular proteins at high levels.
Cleavage by overlap extension by PCR using asymmetric amplification: an improved technique for the generation of hybrid proteins of immunological interest.
