pET Expression System Research Articles

Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.

Characterization of Foot-and-Mouth Disease Virus Serotype O-Specific Single Domain Antibody Expressed in the pET Expression System.

Foot and mouth disease (FMD) is an extremely contagious disease of cloven-hoofed domesticated and wild animals, resulting in significant economic losses in many parts of the world. FMD virus (FMDV) serotype O is responsible for approximately 70% of global outbreaks. For detection of FMDV antigen or antibody, ELISAs are used worldwide and have several limitations, such as batch-to-batch variation in generating immunobiologicals, high production cost and ethical concerns over animal sacrifice. The use of single domain antibody (sdAb) or variable N-terminal domain of the heavy chain of heavy chain antibody (VHH) found naturally in camels has proven their effectiveness in diagnostics and therapeutics. In the present study, the anti-FMDV serotype O-specific VHH-C1 gene sequence (Accession no. KJ751546) was retrieved from the NCBI database. The gene was synthesized commercially in the pBluescript KS+ cloning vector and expressed in E. coli BL21 (DE3) cells using the pET303/CT-His expression system with a C-terminal 6X-His tag. The expressed sdAb, verified by SDS‒PAGE and western blotting, was purified by Ni-chelate chromatography and used as a coating antibody in double antibody sandwich (DAS) ELISA for FMDV detection and typing. The sdAb exhibited a high binding affinity for FMDV serotype O, without any cross-reactivity toward serotypes A and Asia-1. It exhibited better thermostability up to 85°C than conventional rabbit polyclonal anti-FMDV sera. The potential of sdAbs thus produced without sacrificing lab animals could be explored for replacing polyclonal sera in DAS-ELISA as well as for developing biosensors or lateral flow devices for FMDV type O detection.

Assessing the Orthogonality of Phage-Encoded RNA Polymerases for Tailored Synthetic Biology Applications in Pseudomonas Species.

The phage T7 RNA polymerase (RNAP) and lysozyme form the basis of the widely used pET expression system for recombinant expression in the biotechnology field and as a tool in microbial synthetic biology. Attempts to transfer this genetic circuitry from Escherichia coli to non-model bacterial organisms with high potential have been restricted by the cytotoxicity of the T7 RNAP in the receiving hosts. We here explore the diversity of T7-like RNAPs mined directly from Pseudomonas phages for implementation in Pseudomonas species, thus relying on the co-evolution and natural adaptation of the system towards its host. By screening and characterizing different viral transcription machinery using a vector-based system in P. putida., we identified a set of four non-toxic phage RNAPs from phages phi15, PPPL-1, Pf-10, and 67PfluR64PP, showing a broad activity range and orthogonality to each other and the T7 RNAP. In addition, we confirmed the transcription start sites of their predicted promoters and improved the stringency of the phage RNAP expression systems by introducing and optimizing phage lysozymes for RNAP inhibition. This set of viral RNAPs expands the adaption of T7-inspired circuitry towards Pseudomonas species and highlights the potential of mining tailored genetic parts and tools from phages for their non-model host.

THE EFFECT OF H. PYLORI PROTEINS JHP 0290 (FL) AND JHP 0290 ON HOST CELL BEHAVIOR

Objective: This study was designed to investigate the effect of H. Pylori proteins JHP 0290 (FL) and JHP 0290 on host cell response. Materials and Methods: This study was conducted at Khyber Teaching Hospital (KTH), Peshawar from April 2021 to September 2021. H. Pylori genomic DNA (J99 and 26695), pET expression system plasmid (PET21a, 22b & 28b+), kanamycin 50µg/ml and ampicillin 100µg/ml, LB agar plates and LB media, Talon@IMAC resin column were used for studying H. pylori proteins. Genes coding for these proteins were successively cloned into PET21a, PET22b, and PET28b+ expression vectors but no expression was seen with different concentrations of IPTG in BL21(DE3) culture, and expression was prolonged for 4hrs. Results: Only JHP0290 (FL) & JHP0290 (-17a.a) were successively cloned in PET28b+ and expression was induced by using 100µM IPTG in E.coli strain (BL21DE3). Restriction analysis showed the size of JHP0290 (FL & -17a.a) at approximately 550bp and protein size was analyzed on SDS-page which was 20Kda approximately. JHP0290FL protein was found in pellets while JHP0290 (-17a.a) protein was found in pellets & supernatant before being applied to Talon cell through resin column to get purified protein. The study showed a clear band of JHP0290 (FL) on SDS-page with imidazole eluted fraction pooled, while a faint band with a distinct band of JHP0290 (-17a.a) was also seen on SDS-page. Conclusion: These changes are indicative of altered host cell response induced by H.pylori protein JHP0290 suggesting its possible involvement in the development and severity of the disease. Keywords: H.pylori proteins, virulence factor, host cell response, genomic DNA, pathogenicity

Developing a dynamic equilibrium system in Escherichia coli to improve the production of recombinant proteins.

The combination of Escherichia coli BL21 (DE3) and the pET expression system is used extensively for the expression of various recombinant proteins (RPs). However, RP overexpression often introduces a growth burden for the host, especially in the case of toxic proteins. The key to solving this problem is to reduce the host burden associated with protein overproduction, which is often achieved by regulating the expression or activity of T7 RNAP or growth-decoupled systems. However, these strategies mainly relieve or interrupt the robbing of host resources, and do not eliminate other types of host burdens in the production process. In this study, we constructed a production system based on a dynamic equilibrium to precisely relieve the host burden and increase the RP production. The system is composed of three modules, including the overexpression of basic growth-related genes (rRNA, RNAP core enzyme, sigma factors), prediction and overexpression of key proteins using the enzyme-constrained model ec_iECBD_1354, and dynamic regulation of growth-related and key protein expression intensity based on a burden-driven promoter. Using this system, the production of many high-burden proteins, including autolysis protein and E. coli membrane proteins, was increased to varying degrees. Among them, the cytosine transporter protein (CodB) was most significantly improved, with a 4.02-fold higher production compared to the wild strain. This system can effectively reduce the optimizing costs, and is suitable for developing various types of RP expression hosts rapidly. KEY POINTS: • The basic growth-related resources can relieve the host burden from recombinant protein. • The enzyme-constrained model can accurately predict key genes to improve yield. • The expression intensity can be dynamically adjusted with changes in burden.

CRISPR-Based Construction of a BL21 (DE3)-Derived Variant Strain Library to Rapidly Improve Recombinant Protein Production.

Escherichia coli BL21 (DE3) is the most widely used host for recombinant protein expression. However, not every protein can be highly expressed in BL21 (DE3), so individual optimization strategies are often required for different proteins, which is time-consuming and difficult to apply rapidly for industrial production. Constructing more hosts is a good choice to enrich protein expression selection. The expression level of T7 RNAP is the core control node of the pET expression system, so regulating its expression level is an effective way of improving the production of difficult-to-express proteins. Various BL21 (DE3)-derived variant hosts with different translation levels of T7 RNAP could be obtained by changing the ribosomal binding site (RBS) sequences of T7 RNAP in a genome. Here, a BL21 (DE3)-derived variant strain library with different RBS sequences of T7 RNAP was constructed using a base editor and CRISPR-Cas9. Notably, the CRISPR-Cas9 system combined with degenerate primers enabled the construction of an RBS library with 87.5% of the theoretical coverage in single editing, which is more convenient and efficient than the use of a base editor. The expression level of a target gene in the variant strain library ranged from 28 to 220% of the parental strain. Furthermore, a high-throughput host-screening platform for recombinant protein production was constructed, which enabled us to obtain the best expression host for certain target proteins in only 3 days. As a proof of concept, the production of all eight difficult-to-express proteins was greatly improved, including autolytic protein, membrane proteins, antimicrobial peptides, and hardly soluble proteins. Among them, the expression of glucose dehydrogenase in the best host exhibited a 298-fold increase compared to the parental strain. This strategy is simple and effective, requires no advanced equipment, and can be carried out in any laboratory.

Construction of an AI-2 quorum sensing induced heterologous protein expression system in Escherichia coli

BackgroundThe pET expression system based on T7 promoter which is induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) is by far the most commonly used system for production of heterogeneous proteins in Escherichia coli. However, this system was limited by obvious drawbacks including the host toxicity and metabolic burden imposed by the presence of IPTG.MethodsIn this study, we incorporated the autoinducer-2 (AI-2) quorum sensing system to realize autoinduction of the pET expression system. The autoinduction expression vector pXWZ1 was constructed by inserting the lsr promoter regions into the pET28a(+) vector. The expression efficiency of the reporter genes gfpuv and lacZ by the pXWZ1 and pET28a(+) vectors were compared.ResultsThe results showed that the expression levels of the both report genes in the cells transformed with pXWZ1 without any addition of exogenous inducer were higher than that transformed with pET28a(+) vectors by the induction of IPTG.ConclusionThis new auto-induction system will exclude the limitations of the IPTG induction including toxic to host and increasing formation of inclusion body and will become a more economical and convenient tool for recombinant protein expression.

Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin-Antitoxin Systems.

A large number of bacterial toxin–antitoxin (TA) systems have been identified so far and different experimental approaches have been explored to investigate their activity and regulation both in vivo and in vitro. Nonetheless, a common feature of these methods is represented by the difficulty in cell transformation, culturing, and stability of the transformants, due to the expression of highly toxic proteins. Recently, in dealing with the type I Lpt/RNAII and the type II YafQ/DinJ TA systems, we encountered several of these problems that urged us to optimize methodological strategies to study the phenotype of recombinant Escherichia coli host cells. In particular, we have found conditions to tightly repress toxin expression by combining the pET expression system with the E. coli C41(DE3) pLysS strain. To monitor the RNase activity of the YafQ toxin, we developed a fluorescence approach based on Thioflavin-T which fluoresces brightly when complexed with bacterial RNA. Fluorescence microscopy was also applied to reveal loss of membrane integrity associated with the activity of the type I toxin Lpt, by using DAPI and ethidium bromide to selectively stain cells with impaired membrane permeability. We further found that atomic force microscopy can readily be employed to characterize toxin-induced membrane damages.

Induction of Immune Responses by Recombinant PH-1 Domain of Infectious Laryngotracheitis Virus Glycoprotein B in Chickens.

Infectious laryngotracheitis virus (ILTV) is a cause of main respiratory disease of chickens controlled through live attenuated vaccines. To reduce the risk of adverse effects associated with live vaccines, a recombinant vaccine expressing PH-1 domain of viral glycoprotein B was constructed using the pET expression system under isopropylthiogalactoside (IPTG) induction. The potential immunogenicity of recombinant PH-1 (rPH-1) was evaluated in chickens. Eight-week-old specific-pathogen-free chickens were intramuscularly administered two doses of rPH-1, 25 and 50 μg, alone or with a combination of ISA70 adjuvant. The humoral immune responses were determined up to 3 months postvaccination at 2 weeks apart. The T cell proliferation response was determined on day 28 after primary immunization. The vaccinated birds with rPH-1/ISA70 developed higher and constant-specific anti-ILTV enzyme-linked immunosorbent assay (ELISA) antibodies than in those vaccinated with rPH-1 alone. Coinjection of rPH-1 and adjuvant significantly (p < 0.01) increased the T cell proliferation responses. There were no significant differences in eliciting the immune responses in chickens immunized with the higher dose of the antigen than that with the lower dose. The data indicate the immunogenic efficiency of rPH-1 against ILTV. Vaccination with recombinant proteins offers a preventing option to control the ILTV infection and could be a candidate to replace current live vaccines.

Construction of T7-Like Expression System in Pseudomonas putida KT2440 to Enhance the Heterologous Expression Level.

Pseudomonas putida KT2440 has become an attractive chassis for heterologous expression with the development of effective genetic manipulation tools. Improving the level of transcriptional regulation is particularly important for extending the potential of P. putida KT2440 in heterologous expression. Although many strategies have been applied to enhance the heterologous expression level in P. putida KT2440, it was still at a relatively low level. Herein we constructed a T7-like expression system in P. putida KT2440, mimicking the pET expression system in Escherichia coli, which consisted of T7-like RNA polymerase (MmP1) integrated strain and the corresponding expression vector for the heterologous expression enhancement. With the optimization of the insertion site and the copy number of RNA polymerase (RNAP), the relative fluorescence intensity (RFI) of the super-folder green fluorescent protein (sfGFP) was improved by 1.4-fold in MmP1 RNAP integrated strain. The induction point and IPTG concentration were also optimized. This strategy was extended to the gene-reduced strain EM42 and the expression of sfGFP was improved by 2.1-fold. The optimal RNAP integration site was also used for introducing T7 RNAP in P. putida KT2440 and the expression level was enhanced, indicating the generality of the integration site for the T7 expression system. Compared to other inducible expression systems in KT2440, the heterologous expression level of the Mmp1 system and T7 system were more than 2.5 times higher. Furthermore, the 3.6-fold enhanced expression level of a difficult-to-express nicotinate dehydrogenase from Comamonas testosteroni JA1 verified the efficiency of the T7-like expression system in P. putida KT2440. Taken together, we constructed and optimized the T7-like and T7 expression system in P. putida, thus providing a set of applicable chassis and corresponding plasmids to improve recombinant expression level, expecting to be used for difficult-to-express proteins.

A novel global transcriptional perturbation target identified by forward genetics reprograms Vibrio natriegens for improving recombinant protein production.

Vibrio natriegens is known to be the fastest-growing free-living bacterium with the potential to be a novel protein expression system other than Escherichia coli. Seven sampled genes of interest (GOIs) encoding biocatalyst enzymes, including Ochrobactrum anthropi-derived ω-transaminase (OATA), were strongly expressed in E. coli but weakly in V. natriegens using the pET expression system. In this study, we fused the C-terminal of OATA with green fluorescent protein (GFP) and obtained V. natriegens mutants that could increase both protein yield and enzyme activity of OATA as well as the other three GOIs by ultraviolet mutagenesis, fluorescence-activated cell sorting (FACS), and OATA colorimetric assay. Furthermore, next-generation sequencing and strain reconstruction revealed that the Y457 variants in the conserved site of endogenous RNA polymerase (RNAP) β' subunit rpoC are responsible for the increase in recombinant protein yield. We speculated that the mutation of rpoC Y457 may reprogram V. natriegens's innate gene transcription, thereby increasing the copy number of pET plasmids and soluble protein yield of certain GOIs. The increase in GOI expression may partly be attributed to the increase in copy number. In conclusion, GOI-GFP fusion combined with FACS is a powerful tool of forward genetics that can be used to obtain a superior expression chassis. If more high-expression-related targets are found for more GOIs, it would make the construction of next-generation protein expression chassis more time-saving.

Transcriptional network analysis identifies key elements governing the recombinant protein production provoked reprogramming of carbon and energy metabolism in Escherichia coli BL21 (DE3)

AbstractThe impact of recombinant protein production on carbon and energy metabolism in Escherichia coli BL21 (DE3) was studied through transcriptome and proteome analysis of cells induced in carbon‐limited fed‐batch cultures during either fast or slow growth. Production of human basic fibroblast growth factor (pET expression system, T7 promoter) during fast growth leads to a macroscopically observable response classifiable into two consecutive steps: i. apparently unperturbed growth and respiration with concomitant formation of pyruvate and acetate followed by ii. inhibition of growth, respiratory activity and glucose uptake. Down‐regulation of genes involved in sugar and acetate uptake, tricarboxylic acid (TCA) cycle, and respiratory energy generation started already during apparently unperturbed growth with the exceptions of up‐regulated genes encoding the less energy efficient NADH dehydrogenase and terminal oxidases. A transcription factor target gene network analysis revealed that observed changes are mainly attributable to the vanishing influence of the transcription factor CRP‐cAMP but also to a strong down‐regulation of AcrA‐P repressed genes. Moreover, down‐regulation of MalT activated and up‐regulation of PdhR repressed genes contribute among others to the reorganization of the transcriptome. The main drivers were identified as accumulating metabolites, for example, pyruvate, which affect transcription factor activity. The resulting restructured proteome leads to reduced glucose uptake, TCA cycle, and respiratory capacities this way decreasing catabolic carbon breakdown and metabolite accumulation. At slow growth, the production provoked transcriptome rearrangements are more subtle not leading to a macroscopically evident response. In summary, the transcriptomic response towards recombinant gene expression mimics a carbon or nutrient up‐shift response aiming to match catabolic carbon processing with compromised anabolic capacities of induced cells. It is not the reason for growth inhibition and the metabolic burden but the cellular attempt to attenuate the “toxic effect” of recombinant gene expression by reducing carbon catabolism.

Production of a Recombinant Peptide (Lasioglossin LL ΙΙΙ) and Assessment of Antibacterial and Antioxidant Activity

The use of antimicrobial peptides (AMPs) as biopreservative to replace chemical preservatives has become of interest among consumers. Current approaches of production and purification of AMPs are time-consuming and costly and may kill the producing host cells. In this study a recombinant peptide was patterned from Lasioglossin LL ΙΙΙ which is found naturally in the venom of eusocial bee, this recombinant peptide was produced to inhibit food microbial degradation with the least possible costs. The synthesis of this peptide in the periplasmic space of Escherichia coli, has facilitated the production and purification of this additive. The pET based expression system for production of a recombinant form of Lasioglossin LL ΙΙΙ using E. coli as a host was developed. The antibacterial and antioxidant activities of recombinant peptide were determined. A C-terminal poly histidine tag was used to facilitate purification by standard methods using Ni2+ affinity chromatography. The average yield of the recombinant peptide was 0.7 g/L. The findings showed great antimicrobial function of the peptide. The antioxidant activity of the peptide was lower than Butylated hydroxytoluene (BHT) synthesized antioxidant as a common antioxidant but the antioxidant potential of the peptide along with the antimicrobial activity against foodborne pathogens have shown promising benefits of the peptide. These results demonstrated that pET expression system is appropriate for the rapid and simple isolation of recombinant lasioglossin LL ΙΙΙ from E. coli and the recombinant peptide could be used as a natural preservative source. However, further studies need to be carried out to check the effect of this peptide on more microorganisms.

Identification of immunodominant IgE binding epitopes of Der p 24, a major allergen of Dermatophagoides pteronyssinus

BackgroundThe identification of house dust mite (HDM) allergens and epitopes is important for allergy diagnosis and treatment. We sought to identify the Dermatophagoides pteronyssinus group 24 allergen (Der p 24) and to identify its immunodominant IgE epitope(s).MethodsDer p 24 cDNA was cloned and expressed in a pET expression system. The IgE binding activity of purified recombinant (r)Der p 24 was evaluated by western blotting. Truncated Der p 24 proteins and overlapping synthetic polypeptides were subjected to IgE binding assays. Balb/c mice were immunized to investigate IgE epitope induction of IgE production. IgE binding of the 32 N-terminal residues of Der p 24 was compared to other Der p epitopes in enzyme-linked immunosorbent assays and dot blot assays. Human skin prick tests (SPTs) were performed.ResultsWe cloned and expressed Der p 24 cDNA (GenBank accession no. KP893174.1). HDM allergic sera bound rDer p 24 in vitro and 5/10 HDM allergic patients (50%) had positive SPT reactions to rDer p 24. The immunodominant IgE epitope of Der p 24 was localized to the N-terminal 32-residue region, which produced a high specific IgE antibody titer in vivo and promoted mast cell β-hexosaminidase release. The IgE binding activity this N-terminal epitope of Der p 24 was stronger than that of Der p 1 or Der p 2 IgE epitopes.ConclusionsWe identified Der p 24 as a major HDM allergen with strong IgE binding activity via an immunodominant IgE epitope in the N-terminal 32-residue region, which triggers IgE production in vivo. The identified Der p 24 epitope may support HDM allergy diagnosis and treatment.

Time-course and degradation rate of membrane scaffold protein (MSP1D1) during recombinant production

Membrane scaffold proteins (MSPs) are synthetic derivatives of apolipoprotein A-I, a major protein component of human high-density lipoprotein complexes. The most common among these is the variant MSP1D1, which has been in the focus of research on membrane mimetics in the past. As such, the amphipathic MSP1D1 has the ability to self-assemble in the presence of synthetic phospholipids into discoidal nanoparticles, so called nanodiscs. The recombinant production of MSP is exclusively reported using a standard laboratory expression system of the pET family. However, strong variations in both yield and achieved concentration as well as complications related to unspecific degradation are commonly reported. In addition, the time-course of recombinant protein as well as specific protein yields have not yet been quantified conclusively. In this study, the time-course of MSP1D1 concentration was investigated in a standard pET expression system in terms of quantification of production and degradation rates in comparison to a reference protein (eGFP).

Development of a high-yield expression and purification system for platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4. rhPF4 was produced using the pET expression system in the BL21(DE3) strain of Escherichia coli. The system was optimized for protein expression using isopropyl β-D-1-thiogalactopyranoside at different induction temperatures and incubation times. rhPF4 solubility was improved by using different detergents during cell lysis and by purifying with heparin affinity and ion exchange chromatography. Biochemical characteristics of rhPF4 were investigated using mass spectrometry, SDS-PAGE analysis, and gel filtration chromatography and compared to platelet-derived PF4. Antigenic and functional characteristics of rhPF4 were studied using the anti-PF4/heparin EIA and the PF4-SRA. Using this method, we could produce 11.4 ± 0.6 mg of pure rhPF4 per liter of bacterial culture. Absorbance readings from the anti-PF4/heparin EIA using platelet-derived and rhPF4 were highly correlated (n = 194; r = 0.9545, p < 0.0001); and functional release of serotonin in the PF4-SRA induced by anti-PF4/heparin antibodies was similar to either platelet-derived or rhPF4 and heparin (r = 0.9597, p < 0.0001). Our method of rhPF4 production is efficient and does not rely on a source of platelets. The rhPF4 purification method described produces greater yields at a lower cost than other current methods. The application of this method can improve the efficiency of biochemical investigations and HIT diagnostic testing by supplying sufficient amounts of PF4.

Genetic determinant of Bacillus pumilus lipase lethality and its application as positive selection cloning vector in Escherichia coli

Positive selection vectors carry genes that upon expression produce proteins that cause host cell deaths. Insertion of foreign DNA fragments within the ORF of the gene disrupts the lethal effect of the expressed protein. This study described the cloning of Family I.4 Bacillus pumilus lipase gene whose expressed protein is toxic and lethal to Escherichia coli JM109 (DE3) cells. The determinant of toxicity was identified through Error-prone PCR to be the nature of amino acid residue resident at position 28 of the mature lipase protein. The presence of Thr/Ser28 within the mature lipases of B. pumilus and B. licheniformis resulted in lethality to E. coli cells. However, the Thr28Ala or Thr28Gly mutations relieved the lethal phenotype of mature Family I.4 Bacillus lipases. The toxic effect of the expressed mature B. pumilus lipase protein was exploited in the development of a positive selection cloning vector. The B. pumilus lipase gene was synthesised to contain 13 unique silent restriction sites within the ORF, and placed under the regulation of T7 promoter of the pET expression system. Insertional inactivation of the gene's toxic protein was achieved by cloning DNA fragments of different sizes within the designed multiple cloning sites. The toxic effect of the lipase protein was disrupted indicating the potential of the gene for application in suicidal positive selection cloning vectors. The results revealed that protein expression and engineering studies aimed at optimal production of mature Family I.4 Bacillus lipases in E. coli should take into consideration the nature of amino acid 28 resident.

Expression of chitinase gene in BL21 pET system and investigating the biocatalystic performance of chitinase-loaded AlgSep nanocomposite beads

Chitin, a polysaccharide, is abundant in nature and this substrate can be easily hydrolyzed by chitinase. Pharmaceutical and industrial applications of chitinase are considerably noteworthy, therefore in this study, high scale production of Chit36 enzyme was targeted using the E. coli pET expression system. The purified Chit36 enzyme was immobilized in Ca2+-cross linked alginate/sepiolite (AlgSep) nanocomposite beads for improving the catalytic activity and stability of Chit36 enzyme during the biocatalytic process. Immobilized enzymes require optimal conditions different from soluble enzymes. The AlgSep nanocomposite can save spatial structure and activity of the enzyme which is critical for enzyme immobilization. The catalytic activity and specific activity of the Chit36 entrapped in alginate nanocomposite beads were evaluated. Results showed that the activity of immobilized Chit36 in Ca-Alginate sepiolite composites beads (3.10±0.63U/g gel) was higher than that of immobilized Chit36 in Ca-alginate beads (3.95±0.40U/g gel). Also, the specific activity of Chit36 in AlgSep nanocomposite beads (22.9±1.521U/mg protein) was higher than the immobilized Chit36 in sepiolite-free alginate beads (8.52±0.758U/mg protein). The promising results obtained from this study would have beneficial pharmaceutical and industrial applications.

Mechanistic platform knowledge of concomitant sugar uptake in Escherichia coli BL21(DE3) strains

When producing recombinant proteins, the use of Escherichia coli strain BL21(DE3) in combination with the T7-based pET-expression system is often the method of choice. In a recent study we introduced a mechanistic model describing the correlation of the specific glucose uptake rate (qs,glu) and the corresponding maximum specific lactose uptake rate (qs,lac,max) for a pET-based E. coli BL21(DE3) strain producing a single chain variable fragment (scFv). We showed the effect of qs,lac,max on productivity and product location underlining its importance for recombinant protein production. In the present study we investigated the mechanistic qs,glu/qs,lac,max correlation for four pET-based E. coli BL21(DE3) strains producing different recombinant products and thereby proved the mechanistic model to be platform knowledge for E. coli BL21(DE3). However, we found that the model parameters strongly depended on the recombinant product. Driven by this observation we tested different dynamic bioprocess strategies to allow a faster investigation of this mechanistic correlation. In fact, we succeeded and propose an experimental strategy comprising only one batch cultivation, one fed-batch cultivation as well as one dynamic experiment, to reliably determine the mechanistic model for qs,glu/qs,lac,max and get trustworthy model parameters for pET-based E. coli BL21(DE3) strains which are the basis for bioprocess development.

The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-d-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (qs,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (qs,lac) and qs,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying qs,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between qs,lac and qs,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7620-7) contains supplementary material, which is available to authorized users.