-
Scientific Reports Oct 2022Bovine enterokinase light chain (EK) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However,...
Bovine enterokinase light chain (EK) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EK gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by k/K, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased k and T values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site.
Topics: Cattle; Animals; Enteropeptidase; Escherichia coli; Inclusion Bodies; Periplasm; Biological Products; Recombinant Proteins
PubMed: 36271247
DOI: 10.1038/s41598-022-22574-6 -
Biophysical Journal Mar 2022The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose. This repression has been attributed to cAMP receptor protein-mediated...
The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose. This repression has been attributed to cAMP receptor protein-mediated inhibition of lac transcription and EIIA-mediated inhibition of lactose transport (inducer exclusion). The growing evidence against the first mechanism has led to the postulate that the repression is driven by inducer exclusion. Although inducer exclusion reduces the permease activity only 2-fold in fully induced cells, it could be more potent in partially induced cells. Here, we show that even in partially induced cells, inducer exclusion reduces the permease activity no more than 6-fold. Moreover, the repression is so small because these experiments are performed in the presence of chloramphenicol. Indeed, when glucose is added to a culture growing on glycerol and TMG, but no chloramphenicol, lac expression is repressed 900-fold. This repression is primarily due to reversal of the positive feedback loop, i.e., the decline of the intracellular TMG level leads to a lower permease level, which reduces the intracellular TMG level even further. The repression in the absence of chloramphenicol is therefore primarily due to positive feedback, which does not exist during measurements of inducer exclusion.
Topics: Chloramphenicol; Escherichia coli; Glucose; Lac Operon; Lactose; Membrane Transport Proteins
PubMed: 35065916
DOI: 10.1016/j.bpj.2022.01.016 -
The Journal of Nutrition Apr 2021While cancer is common, its incidence varies widely by tissue. These differences are attributable to variable risk factors, such as environmental exposure, genetic...
BACKGROUND
While cancer is common, its incidence varies widely by tissue. These differences are attributable to variable risk factors, such as environmental exposure, genetic inheritance, and lifetime number of stem cell divisions in a tissue. Folate deficiency is generally associated with increased risk for colorectal cancer (CRC) and acute lymphocytic leukemia (ALL). Conversely, high folic acid (FA) intake has also been associated with higher CRC risk.
OBJECTIVE
Our objective was to compare the effect of folate intake on mutant frequency (MF) and types of mutations in the colon and bone marrow of mice.
METHODS
Five-week-old MutaMouse male mice were fed a deficient (0 mg FA/kg), control (2 mg FA/kg), or supplemented (8 mg FA/kg) diet for 20 wk. Tissue MF was assessed using the lacZ mutant assay and comparisons made by 2-factor ANOVA. LacZ mutant plaques were sequenced using next-generation sequencing, and diet-specific mutation profiles within each tissue were compared by Fisher's exact test.
RESULTS
In the colon, the MF was 1.5-fold and 1.3-fold higher in mice fed the supplemented diet compared with mice fed the control (P = 0.001) and deficient (P = 0.008) diets, respectively. This contrasted with the bone marrow MF in the same mice where the MF was 1.7-fold and 1.6-fold higher in mice fed the deficient diet compared with mice fed the control (P = 0.02) and supplemented (P = 0.03) diets, respectively. Mutation profiles and signatures (mutation context) were tissue-specific.
CONCLUSIONS
Our data indicate that dietary folate intake affects mutagenesis in a tissue- and dose-specific manner in mice. Mutation profiles were generally tissue- but not dose-specific, suggesting that altered cellular folate status appears to interact with endogenous mutagenic mechanisms in each tissue to create a permissive context in which specific mutation types accumulate. These data illuminate potential mechanisms underpinning differences in observed associations between folate intake/status and cancer.
Topics: Animals; Bone Marrow; Colon; Dose-Response Relationship, Drug; Folic Acid; Folic Acid Deficiency; High-Throughput Nucleotide Sequencing; Lac Operon; Male; Mice; Mice, Mutant Strains; Mice, Transgenic; Mutagenesis; Mutation Rate; Organ Specificity
PubMed: 33693772
DOI: 10.1093/jn/nxaa402 -
Protein and Peptide Letters 2021Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems....
BACKGROUND
Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level.
OBJECTIVES
In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium.
METHODS
First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression).
RESULTS
Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture.
CONCLUSION
The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.
Topics: Angiotensinogen; Catalase; Cell Culture Techniques; Culture Media; Escherichia coli; Gene Expression; Glucose; Lac Operon; Lactose; Promoter Regions, Genetic; Recombinant Proteins
PubMed: 34353248
DOI: 10.2174/0929866528666210805120715 -
Microbial Cell Factories Mar 2020The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI)...
BACKGROUND
The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences.
RESULTS
We showed that, in genome-integrated E. coli expression systems that used σ promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.
CONCLUSIONS
Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.
Topics: DNA-Directed RNA Polymerases; Escherichia coli; Gene Expression Regulation, Bacterial; Genome, Bacterial; Green Fluorescent Proteins; Lac Operon; Lac Repressors; Promoter Regions, Genetic; Recombinant Proteins; Viral Proteins
PubMed: 32138729
DOI: 10.1186/s12934-020-01311-6 -
Frontiers in Microbiology 2023[This corrects the article DOI: 10.3389/fmicb.2021.709259.].
[This corrects the article DOI: 10.3389/fmicb.2021.709259.].
PubMed: 38088962
DOI: 10.3389/fmicb.2023.1332365 -
MBio Apr 2024We previously demonstrated that mutation of in limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of...
We previously demonstrated that mutation of in limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of these phenotypes can be attributed to the increased production of extracellular proteases. Here we extend these studies to assess the individual importance of these proteases alone and in combination with each other using the methicillin-resistant USA300 strain LAC, the methicillin-susceptible USA200 strain UAMS-1, and isogenic mutants that were also unable to produce aureolysin (Aur), staphopain A (ScpA), staphylococcal serine protease A (subsp.), staphopain B (SspB), and the staphylococcal serine protease-like proteins A-F (SplA-F). Biofilm formation was restored in LAC and UAMS-1 mutants by subsequent mutation of and , while mutation of had the greatest impact on cytotoxicity to mammalian cells, particularly with conditioned medium (CM) from the more cytotoxic strain LAC. However, SDS-PAGE and western blot analysis of CM confirmed that mutation of was also required to mimic the phenotype of mutants unable to produce any extracellular proteases. Nevertheless, in a murine model of post-traumatic osteomyelitis, mutation of and had the greatest impact on restoring the virulence of LAC and UAMS-1 mutants, with concurrent mutation of and the operon having relatively little effect. These results demonstrate that the increased production of Aur and ScpA in combination with each other is a primary determinant of the reduced virulence of mutants in diverse clinical isolates including both methicillin-resistant and methicillin-susceptible strains.IMPORTANCEPrevious work established that SarA plays a primary role in limiting the production of extracellular proteases to prevent them from limiting the abundance of virulence factors. Eliminating the production of all 10 extracellular proteases in the methicillin-resistant strain LAC has also been shown to enhance virulence in a murine sepsis model, and this has been attributed to the specific proteases Aur and ScpA. The importance of this work lies in our demonstration that the increased production of these same proteases largely accounts for the decreased virulence of mutants in a murine model of post-traumatic osteomyelitis not only in LAC but also in the methicillin-susceptible human osteomyelitis isolate UAMS-1. This confirms that -mediated repression of Aur and ScpA production plays a critical role in the posttranslational regulation of virulence factors in diverse clinical isolates and diverse forms of infection.
Topics: Animals; Mice; Humans; Staphylococcus aureus; Virulence; Disease Models, Animal; Methicillin; Virulence Factors; Bacterial Proteins; Peptide Hydrolases; Osteomyelitis; Biofilms; Staphylococcal Infections; Gene Expression Regulation, Bacterial; Mammals; Metalloendopeptidases
PubMed: 38415646
DOI: 10.1128/mbio.03383-23 -
Molecular Microbiology Jan 2021Lactose is an abundant dietary carbohydrate metabolized by the dental pathogen Streptococcus mutans. Lactose metabolism presents both classic diauxic behaviors and...
Lactose is an abundant dietary carbohydrate metabolized by the dental pathogen Streptococcus mutans. Lactose metabolism presents both classic diauxic behaviors and long-term memory, where the bacteria can pause for >11 h before initiating growth on lactose. Here, we explored mechanisms contributing to unusual aspects of regulation of the lac operon. The fructose-phosphate metabolites, F-1-P and F-6-P, could modulate the DNA-binding activities of the lactose repressor. Recombinant LacR proteins bound upstream of lacA and Gal-6-P induced the formation of different LacR-DNA complexes. Deletion of lacR resulted in strain-specific growth phenotypes on lactose, but also on a number of mono- and di-saccharides that involve the glucose-PTS or glucokinase in their catabolism. The phenotypes were consistent with the novel findings that loss of LacR altered glucose-PTS activity and expression of the gene for glucokinase. CcpA was also shown to affect lactose metabolism in vivo and to bind to the lacA promoter region in vitro. Collectively, our study reveals complex molecular circuits controlling lactose metabolism in S. mutans, where LacR and CcpA integrate cellular and environmental cues to regulate metabolism of a variety of carbohydrates that are critical to persistence and pathogenicity of S. mutans.
Topics: Bacterial Proteins; Carbohydrate Metabolism; Catabolite Repression; Fructose; Galactose; Gene Expression; Gene Expression Regulation, Bacterial; Genes, Bacterial; Glucose; Lac Operon; Lactose; Operon; Promoter Regions, Genetic; Streptococcus mutans
PubMed: 32881130
DOI: 10.1111/mmi.14597 -
Frontiers in Molecular Biosciences 2022GAL network in the yeast is one of the most well-characterized regulatory network. Expression of GAL genes is contingent on exposure to galactose, and an appropriate...
GAL network in the yeast is one of the most well-characterized regulatory network. Expression of GAL genes is contingent on exposure to galactose, and an appropriate combination of the alleles of the regulatory genes GAL3, GAL1, GAL80, and GAL4. The presence of multiple regulators in the GAL network makes it unique, as compared to the many sugar utilization networks studied in bacteria. For example, utilization of lactose is controlled by a single regulator LacI, in 's lac operon. Moreover, recent work has demonstrated that multiple alleles of these regulatory proteins are present in yeast isolated from ecological niches. In this work, we develop a mathematical model, and demonstrate via deterministic and stochastic runs of the model, that behavior/gene expression patterns of the cells (at a population level, and at a single-cell resolution) can be modulated by altering the binding affinities between the regulatory proteins. This adaptability is likely the key to explaining the multiple GAL regulatory alleles discovered in ecological isolates in recent years.
PubMed: 35372523
DOI: 10.3389/fmolb.2022.801011 -
Microbial Biotechnology Mar 2024Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction...
Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the P /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS).
Topics: Lac Repressors; Escherichia coli; Escherichia coli Proteins; Mutation; Promoter Regions, Genetic; Bacterial Proteins
PubMed: 38465475
DOI: 10.1111/1751-7915.14427