Labeling of methyl groups: a streamlined protocol and guidance for the selection of 2H precursors based on molecular weight.

A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2O and 2H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D2O) or deuterated glucose is not required to obtain 1H-13C correlated NMR spectra of a 50 kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100-150 kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the ß-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.

A multidomain PARP14 construct suitable for bacterial expression.

Poly-ADP-ribose polymerase-14 (PARP14) can modify proteins and nucleic acids by the reversible addition of a single ADP-ribose molecule. Aberrant PARP14 functions have been related to cancer and inflammation, and its domains are involved in processes related to viral infection. Previous research indicates that PARP14 functions might be mediated via a multitude of target proteins. In vitro studies of this large multidomain enzyme have been complicated by difficulties to obtain biochemical quantities of pure protein. Here we present a strategy that allows bacterial expression and purification of a functional multidomain construct of PARP14. We substituted an internal KH domain and its neighboring unstructured region with a SUMO domain to obtain a protein construct that encompasses three macrodomains, a WWE domain, and a PARP catalytic domain. We show that the resulting construct retains both ADP-ribosyltransferase and de-MARylase activities. This construct will be useful in structural and functional studies of PARP14.

Expression, purification, and characterization of transmembrane protein homogentisate solanesyltransferase.

Homogentisate solanesyltransferase (HST) is a crucial enzyme in the plastoquinone biosynthetic pathway and has recently emerged as a promising target for herbicides. In this study, we successfully expressed and purified a stable and highly pure form of seven times transmembrane protein Chlamydomonas reinhardtii HST (CrHST). The final yield of CrHST protein obtained was 12.2 mg per liter of M9 medium. We evaluated the inhibitory effect on CrHST using Des-Morpholinocarbony Cyclopyrimorate (DMC) and found its IC50 value to be 3.63 ± 0.53 µM, indicating significant inhibitory potential. Additionally, we investigated the substrate affinity of CrHST with two substrates, determining the Km values as 22.76 ± 1.70 µM for FPP and 48.54 ± 3.89 µM for HGA. Through sequence alignment analyses and three-dimensional structure predictions, we identified conserved amino acid residues forming the active cavity in the enzyme. The results from molecular docking and binding energy calculations indicate that DMC has a greater binding affinity with HST compared to HGA. These findings represent substantial progress in understanding CrHST's properties and potential for herbicide development. KEY POINTS: • First high-yield transmembrane CrHST protein via E. coli system • Preliminarily identified active cavity composition via activity testing • Determined substrate and inhibitor modes via molecular docking.

Unique Behavior of Bacterially Expressed Rat Carnitine Palmitoyltransferase 2 and Its Catalytic Activity.

Mammalian type 2 carnitine parmitoyltransferase (EC 2.3.1.21), abbreviated as CPT2, is an enzyme involved in the translocation of fatty acid into the mitochondrial matrix space, and catalyzes the reaction acylcarnitine + CoA = acyl-CoA + carnitine. When rat CPT2 was expressed in Escherichia coli, its behavior was dependent on the presence or absence of i) its mitochondrial localization sequence and ii) a short amino acid sequence thought to anchor it to the mitochondrial inner membrane: CPT2 containing both sequences behaved as a hydrophobic protein, while recombinant CPT2 lacking both regions behaved as a water soluble protein; if only one region was present, the resultant proteins were observed in both fractions. Because relatively few protein species could be obtained from bacterial lysates as insoluble pellets under the experimental conditions used, selective enrichment of recombinant CPT2 protein containing both hydrophobic sequences was easily achieved. Furthermore, when CPT2 enriched in insoluble fraction was resuspended in an appropriate medium, it showed catalytic activity typical of CPT2: it was completely suppressed by the CPT2 inhibitor, ST1326, but not by the CPT1 inhibitor, malonyl-CoA. Therefore, we conclude that the bacterial expression system is an effective tool for characterization studies of mammalian CPT2.

Co-expression of different proteins in Escherichia coli using plasmids with identical origins of replication.

It is generally accepted that the use of two different plasmids with the identical origins of replication in bacteria is not desirable due to their "incompatibility". The utilization of the same bacterial enzymatic apparatus for replication of different plasmids is thought to cause a significant redistribution in favor of one of them. In the present work, examining co-expression of two different fluorescent proteins in Escherichia coli, we have shown that the use of highly homologous plasmids with identical origins of replication and providing resistance to different antibiotics results in high representation of both plasmids in bacteria. Meanwhile, the level of gene expression and the amount of proteins produced may differ and is determined mostly by their sequence rather than by the "incompatibility" of the plasmids.

Recent Developments in Bioprocessing of Recombinant Antibody Fragments.

Biotechnological and biomedical applications of antibodies have been on a steady rise since the 1980s. As unique and highly specific bioreagents, monoclonal antibodies (mAbs) have been widely exploited and approved as therapeutic agents. However, the use of mAbs has limitations for therapeutic applications. Antibody fragments (AbFs) with preserved antigen-binding sites have a significant potential to overcome the disadvantages of conventional mAbs, such as heterogeneous tissue distribution after systemic administration, especially in solid tumors, and Fc-mediated bystander activation of the immune system. AbFs possess better biodistribution coefficient due to lower molecular weight. They preserve the functional features of mAbs, such as antigen specificity and binding, while at the same time, ensuring much better tissue penetration. An additional benefit of AbFs is the possibility of their production in bacterial and yeast cells due to the small size, more robust structure, and lack of posttranslational modifications. In this review, we described current approaches to the AbF production with recent examples of AbF synthesis in bacterial and yeast expression systems and methods for the production optimization.

Facile purification of active recombinant mouse cytosolic carboxypeptidase 6 from Escherichia coli.

CCP6 is a member of cytosolic carboxypeptidases (CCPs) family, an eraser of a reversible protein posttranslational modification - polyglutamylation, and represents a potential therapeutic target. Currently, production of CCPs mainly depends on eukaryotic expression system, which is time-consuming and costly. Here, we reported that mouse origin full-length CCP6 can be successfully expressed in the soluble fraction of bacteria ArcticExpress (DE3) strain. However, the recombinant mCCP6 was initially co-purified with Cpn60 in a stoichiometric ratio of roughly 1:7 and exhibited no enzyme activity. When coupled with a step to promote the release of the substrate protein from the chaperonins by treatment with ATP/Mg2+/K+, the recombinant CCP6 with deglutamylation activity was obtained, though still partially associated with Cpn60. This is the first report, to our knowledge, that the successful expression and purification of active recombinant mammalian CCPs using a bacterial system was achieved.

Expression of SARS-CoV-2 surface glycoprotein fragment 319-640 in E. coli, and its refolding and purification.

Sensitive and specific serology tests are essential for epidemiological and public health studies of COVID-19 and for vaccine efficacy testing. The presence of antibodies to SARS-CoV-2 surface glycoprotein (Spike) and, specifically, its receptor-binding domain (RBD) correlates with inhibition of SARS-CoV-2 binding to the cellular receptor and viral entry into the cells. Serology tests that detect antibodies targeting RBD have high potential to predict COVID-19 immunity and to accurately determine the extent of the vaccine-induced immune response. Cost-effective methods of expression and purification of Spike and its fragments that preserve antigenic properties are essential for development of such tests. Here we describe a method of production of His6-tagged S319-640 fragment containing RBD in E. coli. It includes expression of the fragment, solubilization of inclusion bodies, and on-the-column refolding. The antigenic properties of the resulting product are similar, but not identical to the RBD-containing fragment expressed in human cells.

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens.

Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of 'difficult-to-express' bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.'s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1-15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.

Bacterial expression and characterization of an anti-CD22 single-chain antibody fragment.

Single-chain variable fragment (scFv) antibodies are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. They possess unique and superior features compared to whole antibodies for immunotherapy of various carcinomas, including hematologic B-cell malignancies. In the presented study we obtained efficient production of the recombinant anti-CD22 scFv in Escherichia coli expression system. The active recombinant protein was successfully recovered from inclusion bodies. Assays were performed to assess the in vitro targeting properties and specificity of the obtained anti-CD22 scFv antibody in the CD22 positive and negative lymphoma cell lines.

Characterization of a hyperphosphorylated variant of G protein-coupled receptor kinase 5 expressed in E. coli.

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in humans and regulate numerous physiological processes through the activation of heterotrimeric G proteins. GPCR kinases (GRKs) selectively phosphorylate active GPCRs, which promotes arrestin binding, receptor internalization, and initiation of alternative signaling pathways. GRK5 is a representative member of one of three GRK subfamilies that does not need post-translational lipidation or other binding partners to exhibit full activity against GPCRs, rendering it a useful tool for biophysical studies directed at characterizing GRK function. However, recombinant expression of GRK5 has thus far been limited to insect and mammalian systems. Here, we describe the expression of functional GRK5 in E. coli and its purification and biochemical characterization. Bacterially expressed GRK5 is hyperphosphorylated, primarily in regions known to be flexible from prior crystal structures, which slightly decreases its catalytic activity toward receptor substrates. Mutation of a single phosphorylation site, Thr10, restores kinetic parameters to those of GRK5 purified from insect cells. Consequently, bacterial expression will allow for production of GRK5 at a reduced cost and faster pace and would facilitate production of isotopically labeled kinase for NMR studies or for the incorporation of unnatural amino acids.

Expression, purification and crystallization of CLK1 kinase - A potential target for antiviral therapy.

Cdc-like kinase 1 (CLK1) is a dual-specificity kinase capable of autophosphorylation on tyrosine residues and Ser/Thr phosphorylation of its substrates. CLK1 belongs to the CLK kinase family that regulates alternative splicing through phosphorylation of serine-arginine rich (SR) proteins. Recent studies have demonstrated that CLK1 has an important role in the replication of influenza A and chikungunya viruses. Furthermore, CLK1 was found to be relevant for the replication of HIV-1 and the West Nile virus, making CLK1 an interesting cellular candidate for the development of a host-directed antiviral therapy that might be efficient for treatment of newly emerging viruses. We describe here our attempts and detailed procedures to obtain the recombinant kinase domain of CLK1 in suitable amounts for crystallization in complex with specific inhibitors. The key solution for the reproducibility of crystals resides in devising and refining expression and purification protocols leading to homogeneous protein. Co-expression of CLK1 with λ-phosphatase and careful purification has yielded crystals of CLK1 complexed with the KH-CB19 inhibitor that diffracted to 1.65 Å. These results paved the path to the screening of more structures of CLK1 complexed compounds, leading to further optimization of their inhibitory activity. Moreover, since kinases are desired targets in numerous pathologies, the approach we report here, the co-expression of kinases with λ-phosphatase, previously used in other kinases, can be adopted as a general protocol in numerous kinase targets for obtaining reproducible and homogenic non-phosphorylated (inactive) forms suitable for biochemical and structural studies thus facilitating the development of novel inhibitors.

Expression, purification, and characterization of the native intracellular domain of human epidermal growth factor receptors 1 and 2 in Escherichia coli.

Human epidermal growth factor receptors (EGFR) are an important target in drug discovery in terms of both protein-small-molecule interactions and protein-protein interactions. In this work, the isolation of a stable soluble protein of the tyrosine kinase domain of EGFR in Escherichia coli expression has been accomplished. This successful study presents the expression and purification conditions to obtain a stable soluble protein of the active tyrosine kinase domain of EGFR (EGFR-TK) and ErbB2 (ErbB2-TK) in a bacterial system, albeit in relatively low yields. The recombinant gene was inserted into a pColdI vector and recombinant protein was expressed at low temperature. Purification of EGFR-TK and ErbB2-TK took place under the same conditions by purified supernatant using a diethylaminoethyl sepharose column followed by anion exchange and size-exclusion chromatography columns. The final yields of purified EGFR-TK and ErbB2-TK were 8.4 and 9.5 mg per liter of culture, respectively. Determination of EGFR-TK and ErbB2-TK was performed via enzyme activity with commercial drugs. The IC50 values of erlotinib and afatinib against EGFR-TK were 13.09 nM and 2.36 nM respectively, while the IC50 values of lapatinib and afatinib against ErbB2-TK were 24.69 nM and 1.36 nM, respectively. These results confirmed that soluble proteins of the active intracellular domain of the HERs family were successfully expressed and purified in a bacterial system. The new protein expression and purification protocol will greatly facilitate the enzymatic inhibition and structural studies of this protein for drug discovery.

Assessment of NS1 protein as an early diagnostic marker for Kyasanur forest disease virus.

BACKGROUND & OBJECTIVES: Due to the emergence of Kyasanur forest disease (KFD) virus to new regions in India, there is an urgent need to develop an early diagnostic system, which is cost-effective and can be efficiently used with minimum paraphernalia. The non-structural-1 (NS1) protein is known to be an early diagnostic marker for flaviviruses. Furthermore, NS1 antigen capture ELISA kits developed using bacterially expressed dengue NS1 protein are commercially available. METHODS: Based on the data available on dengue virus, West Nile virus and other flaviviruses, bacterially expressed Kyasanur forest disease virus (KFDV) NS1 protein and polyclonal serum raised against the NS1 protein in mice and rabbit were used to develop an antigen capture ELISA for early diagnosis of the virus. The feasibility of this ELISA was further tested using in silico predictions. RESULTS: KFDV NS1 gene was cloned, expressed and confirmed by SDS-PAGE and western blotting. An antigen detection ELISA was standardized and sensitivity and specificity was tested with other flaviviruses. KFDV acute phase 43 samples were tested and only two were found to be positive for KFDV NS1 antigen. Superimposition of KFDV NS1 and TBEV NS1 revealed a root mean square distance (RMSD) of ~0.79 Å covering 1220 backbone atoms. This implies that the structures are very similar in terms of 3D fold. The identity of amino acid composition between these proteins was 73.4% and similarity was 92.9%, as revealed from the pairwise comparison. INTERPRETATION & CONCLUSION: The study points out that the half-life, expression and secretion levels of KFDV NS1 protein are not sufficient enough for its use as early diagnostic marker. The protein may have to be expressed in eukaryotic host to counter the lack of glycosylation in bacterial plasmid based expression of proteins. Hence, bacterially expressed KFDV NS1 protein may not be an ideal early diagnostic marker for the virus.

Effects of recombinant salmon type II growth hormone and bovine growth hormone on growth of coho salmon (Oncorhynchus kisutch).

A comparison of the efficacy of salmon and bovine growth hormone to stimulate growth of coho salmon juveniles was performed. Oncorhynchus nerka (sockeye salmon) type II growth hormone (nGH2) was produced using a bacterial expression system, yielding approximately 25 mg of refolded recombinant protein per litre of cells. The purified nGH2 and bovine growth hormone (bGH) were tested in juvenile O. kisutch (coho salmon) over 24 weeks. Weekly intraperitoneal injections of 0.1 and 0.5 µg/g nGH2 resulted in a dose-dependent increase in weight and fork length compared to control fish injected with bovine serum albumin (BSA). Application of 0.5 µg/g bGH resulted in the same stimulation of growth as did 0.5 µg/g nGH2, indicating these proteins were equipotent. Following 6 weeks of treatment and a subsequent rest period of 7 weeks, coho salmon were further treated with bGH at 0.5 µg/g. A prior treatment with bGH did not reduce growth-promoting activity of bGH in subsequent treatments. Throughout the experiment, condition factor decreased at similar rates for all treatment groups. These data show that bGH, which is widely available, can be used to elevate growth rate in juvenile salmon comparably to homologous GH, and validate the use of bGH in physiological or ecological experiments where rapid growth is desired compared to that seen in wild type.

Characterisation of the Carboxypeptidase G2 Catalytic Site and Design of New Inhibitors for Cancer Therapy.

The enzyme carboxypeptidase G2 (CPG2) is used in antibody-directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small-molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high-affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment.

UbiGate: a synthetic biology toolbox to analyse ubiquitination.

Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post-translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process. Here, we present UbiGate - a synthetic biology toolbox, together with an inducible bacterial expression system - to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by 'Golden Gate' cloning. This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression. We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time- and cost-effective manner.

Bacterial expression, purification and biophysical characterization of the smallest plant reticulon isoform, RTNLB13.

Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in length but all contain a conserved C-terminal reticulon homology domain (RHD) that associates with membranes. An understanding of the structure and membrane interactions of RHDs is key to unlocking their mechanism of function, however no three-dimensional structure has been solved. We believe that this is, in part, due to difficulties in obtaining reticulon proteins in yields sufficient for structural study. To address this, we report here the first bacterial overexpression, purification, and biophysical investigation of a reticulon protein from plants, the RTNLB13 protein from A. thaliana. RTNLB13 is the smallest plant reticulon and is made up of a single RHD. We used circular dichroism, SDS-PAGE and analytical ultracentrifugation to reveal that RTNLB13 is 45% α-helical in a number of detergent environments, monomeric at low concentrations, and capable of self-association at higher concentrations. We used solution-state NMR to screen the effect of detergent type on the fold of isotopically-enriched RTNLB13, and found that ∼60% of the expected protein peaks were broadened due to slow dynamics. This broadening points toward a large network of protein-membrane interactions throughout the sequence. We have interpreted our results in light of current literature and suggest a preliminary description of RTNLB13 structure and topology.

The workability of Escherichia coli BL21 (DE3) and Pseudomonas putida KT2440 expression platforms with autodisplayed cellulases: a comparison.

This article comparatively reports the workability of Escherichia coli BL21(DE3) and Pseudomonas putida KT2440 cell factories for the expression of three model autodisplayed cellulases (i.e., endoglucanase, BsCel5A; exoglucanase, CelK; ß-glucosidase, BglA). The differentiation of the recombinant cells was restricted to their cell growth and enzyme expression/activity attributes. Comparatively, the recombinant E. coli showed higher cell growth rates but lower enzyme activities than the recombinant P. putida. However, the endo-, exoglucanase, and ß-glucosidase on the surfaces of both cell factories showed activity over a broad range of pH (4-10) and temperature (30-100 °C). The pH and temperature optima were pH 6, 60 °C (BsCel5A); pH 6, 60-70 °C (CelK); and pH 6, 50 °C (BglA). Overall, the P. putida cell factory with autodisplayed enzymes demonstrated higher bioactivity and remarkable biochemical characteristics and thus was chosen for the saccharification of filter paper. A volumetric blend of the three cellulases with P. putida as the host yielded a ratio of 1:1:1.5 of endoglucanase, exoglucanase, and ß-glucosidase, respectively, as the optimum blend composition for filter paper degradation. At an optical density (578 nm) of 50, the blend generated a maximum sugar yield of about 0.7 mg/ml (~ 0.08 U/g) from Whatman filter paper (Ø 6 mm, ~ 2.5 mg) within 24 h.

The catalytic activity of a recombinant single chain variable fragment nucleic acid-hydrolysing antibody varies with fusion tag and expression host.

The antigen-binding properties of single chain Fv antibodies (scFvs) can vary depending on the position and type of fusion tag used, as well as the host cells used for expression. The issue is even more complicated with a catalytic scFv antibody that binds and hydrolyses a specific antigen. Herein, we investigated the antigen-binding and -hydrolysing activities of the catalytic anti-nucleic acid antibody 3D8 scFv expressed in Escherichia coli or HEK293f cells with or without additional amino acid residues at the N- and C-termini. DNA-binding activity was retained in all recombinant forms. However, the DNA-hydrolysing activity varied drastically between forms. The DNA-hydrolysing activity of E. coli-derived 3D8 scFvs was not affected by the presence of a C-terminal human influenza haemagglutinin (HA) or His tag. By contrast, the activity of HEK293f-derived 3D8 scFvs was completely lost when additional residues were included at the N-terminus and/or when a His tag was incorporated at the C-terminus, whereas a HA tag at the C-terminus did not diminish activity. Thus, we demonstrate that the antigen-binding and catalytic activities of a catalytic antibody can be separately affected by the presence of additional residues at the N- and C-termini, and by the host cell type.