Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells

Background

Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.?

Methods

Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the E?xpi293 cells with PEI (which has a negligible cost).

Results

The protocol has been used to generate multiple variants of tetra- and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (Aβ) aggregates in vivo.

Conclusions

This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.

     

Copper oxide nanoparticles induce anticancer activity in A549 lung cancer cells by inhibition of histone deacetylase

Objectives

Copper oxide nanoparticles (CuO NPs) promoting anticancer activity may be due to the regulation of various classes of histone deacetylases (HDACs).

Results

Green-synthesized CuO NPs significantly arrested total HDAC level and also suppressed class I, II and IV HDACs mRNA expression in A549 cells. A549 cells treated with CuO NPs downregulated oncogenes and upregulated tumor suppressor protein expression. CuO NPs positively regulated both mitochondrial and death receptor-mediated apoptosis caspase cascade pathway in A549 cells.

Conclusion

Green-synthesized CuO NPs inhibited HDAC and therefore shown apoptosis mediated anticancer activity in A549 lung cancer cell line.

     

A tightly regulated expression system for <Emphasis Type="Italic">E. coli</Emphasis> using supersaturated silicic acid

Objective

To develop a new expression system regulated by a ferric uptake regulator in which silicic acid is used as an inducer.

Results

Fur box (binding site for Fur) was substituted for the lac operator to regulate the expression of GFP with the lac promoter. Since the addition of supersaturated silicic acid invokes iron deficiency, supersaturated silicic acids were used as an inducer. GFP expression was dependent on silica concentration, and the expression level without silica was negligible. Basal expression level of this lac-Fur system was extremely low and, hence, lytic enzyme gene E from bacteriophage ?X174 could be retained in this system. Furthermore, the expression of genes of interest was spontaneously initiated as the cell density increased and the costs of the inducer are considerably less than IPTG.

Conclusion

The combination of lac promoter and Ferric uptake repressor allowed the protein expression by supersaturated silicic acid as an inducer in an easy and cost-effective way.

     

UHRF2 promotes DNA damage response by decreasing p21 via RING finger domain

Objectives

To investigate the interaction of E3 ubiquitin ligase UHRF2 with p21 and the mechanism of UHRF2 in repairing DNA damage caused by hydroxyurea (HU) in HEK293 cells.

Results

Western blotting indicated that the overexpression of UHRF2 reduced the level of p21, particularly in HEK293 cells. Immunoprecipitation and immunofluorescence staining reveled that UHRF2 combined with p21 in the nucleus. In addition, UHRF2 degraded p21 through ubiquitination and shortened the half-life of p21. UHRF2 could repair DNA damage caused by HU treatment, which was impaired by the inhibition of p21 in HEK293 cells.

Conclusions

UHRF2 may negatively modulate p21 to regulate DNA damage response, suggesting a novel pathway of UHRF2 repairing DNA damage through the partial regulation of p21.

     

Stability of targeted metabolite profiles of urine samples under different storage conditions

Introduction

Few studies have investigated the influence of storage conditions on urine samples and none of them used targeted mass spectrometry (MS).

Objectives

We investigated the stability of metabolite profiles in urine samples under different storage conditions using targeted metabolomics.

Methods

Pooled, fasting urine samples were collected and stored at ?80 °C (biobank standard), ?20 °C (freezer), 4 °C (fridge), ~9 °C (cool pack), and ~20 °C (room temperature) for 0, 2, 8 and 24 h. Metabolite concentrations were quantified with MS using the AbsoluteIDQ? p150 assay. We used the Welch-Satterthwaite-test to compare the concentrations of each metabolite. Mixed effects linear regression was used to assess the influence of the interaction of storage time and temperature.

Results

The concentrations of 63 investigated metabolites were stable at ?20 and 4 °C for up to 24 h when compared to samples immediately stored at ?80 °C. When stored at ~9 °C for 24 h, few amino acids (Arg, Val and Leu/Ile) significantly decreased by 40% in concentration (P < 7.9E?04); for an additional three metabolites (Ser, Met, Hexose H1) when stored at ~20 °C reduced up to 60% in concentrations. The concentrations of four more metabolites (Glu, Phe, Pro, and Thr) were found to be significantly influenced when considering the interaction between exposure time and temperature.

Conclusion

Our findings indicate that 78% of quantified metabolites were stable for all examined storage conditions. Particularly, some amino acid concentrations were sensitive to changes after prolonged storage at room temperature. Shipping or storing urine samples on cool packs or at room temperature for more than 8 h and multiple numbers of freeze and thaw cycles should be avoided.

     

Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX

Objectives

To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection.

Results

Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells.

Conclusion

Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.

     

Delivery of HIV-1 Nef Protein in Mammalian Cells Using Cell Penetrating Peptides as a Candidate Therapeutic Vaccine

The HIV-1 Nef protein expressed early in viral life cycle has been known as a potent candidate for therapeutic vaccine development. Due to different cell barriers, various cell penetrating peptides (CPPs) such as Pep-1 and CADY-2 have been known to deliver biologically active proteins to cytoplasmic compartments via the plasma membrane. In current study, we firstly evaluated the efficiency of lentiviral vector (pCDH-CMV-MCS-EF1-cGFP-T2A-puro) and eukaryotic expression vector (pEGFP-N1) for expression of HIV-1 Nef protein in HEK-293T cells using TurboFect transfection reagent. Our results showed that both vectors can effectively express the Nef proteins within the target cell. The pEGFP-N1 was more effective than pCDH-GFP for protein expression. Furthermore, Nef protein was expressed in E. coli as GST-Nef fusion and transfected by the amphipathic CPPs including Pep-1 and CADY-2 into HEK-293T cells. The size and morphology of the GST-Nef/CPP complexes were evaluated by scanning electron microscopy, and Zetasizer. Our data indicated that the recombinant GST-Nef protein generated in BL21 strain migrated as a clear band of ~50 kDa in SDS-PAGE. The CPP/GST-Nef nanoparticles were formed with a diameter of below 200 nm and notably delivered into HEK-293T cells. Generally, the Nef protein was expressed in prokaryotic and eukaryotic expression systems using different vectors and efficiently transfected in mammalian cells using various delivery systems. The in vitro efficient delivery of HIV-1 Nef gene and also its protein supports the potential of Nef DNA constructs and CPPs as potent carriers of Nef protein for HIV vaccine design in Future.     

Metabolic signatures of four major histological types of lung cancer cells

Introduction

Histologically lung cancer is classified into four major types: adenocarcinoma (Ad), squamous cell carcinoma (SqCC), large cell carcinoma (LCC), and small cell lung cancer (SCLC). Presently, our understanding of cellular metabolism among them is still not clear.

Objectives

The goal of this study was to assess the cellular metabolic profiles across these four types of lung cancer using an untargeted metabolomics approach.

Methods

Six lung cancer cell lines, viz., Ad (A549 and HCC827), SqCC (NCl-H226 and NCl-H520), LCC (NCl-H460), and SCLC (NCl-H526), were analyzed using liquid chromatography quadrupole time-of-flight mass spectrometry, with normal human small airway epithelial cells (SAEC) as the control group. The principal component analysis (PCA) was performed to identify the metabolic signatures that had characteristic alterations in each histological type. Further, a metabolite set enrichment analysis was performed for pathway analysis.

Results

Compared to the SAEC, 31, 27, 34, 34, 32, and 39 differential metabolites mainly in relation to nucleotides, amino acid, and fatty acid metabolism were identified in A549, HCC827, NCl-H226, NCl-H520, NCl-H460, and NCl-H526 cells, respectively. The metabolic signatures allowed the six cancerous cell lines to be clearly separated in a PCA score plot.

Conclusion

The metabolic signatures are unique to each histological type, and appeared to be related to their cell-of-origin and mutation status. The changes are useful for assessing the metabolic characteristics of lung cancer, and offer potential for the establishment of novel diagnostic tools for different origin and oncogenic mutation of lung cancer.

     

High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Background

Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure.

Results

Transfection conditions were first optimized to achieve expression levels between 10 and 18?mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4–9?mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (K_d) of 5?nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured k_cat (7.2?±?0.5?min^??1) and K_M (1.2?±?0.3?μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption.

Conclusions

The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher k_cat and K_M values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway.

     

An efficient rapid system for assaying HBx-mediated transactivation

Objectives

To develop a rapid and accurate assay system for screening inhibitors or enhancing agents targeting the transactivation capability of hepatitis B virus X protein (HBx) that activates cellular promoters in host cells to facilitate viral replication.

Results

We constructed a new GFP-based reporter system which was different from a luciferase-based reporter system. Firstly, a FLAG-tagged HBx gene was inserted into an expression plasmid, resulting in plasmid pHBx. Next, HBx-FLAG was linked to EGFP by the internal ribosome entry site resulting in plasmid pHBxE. The transactivation effect of HBx-flag on cytomegalovirus (CMV) promoter was verified by EGFP expression using fluorescence quantitation and qPCR. Furthermore, the transactivation ability of the HBx gene was quantified by flow cytometry. Finally, this assay system was tested by known regulators of HBx including DDB1, ID1, and P53. As expected, the GFP reporter level in 293T cells changed with the increasing of HBx regulators. Furthermore, the system modeling the function of transactivation repressor in Hep3B, a HBV-integrated cell line.

Conclusion

Collectively, the GFP-based reporter system provides a rapid and accurate approach for analyzing transactivation ability of HBx.

     

Mutations in human lipoyltransferase gene <Emphasis Type="Italic">LIPT1</Emphasis>cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase

Background

Synthesis and apoenzyme attachment of lipoic acid have emerged as a new complex metabolic pathway. Mutations in several genes involved in the lipoic acid de novo pathway have recently been described (i.e., LIAS, NFU1, BOLA3, IBA57), but no mutation was found so far in genes involved in the specific process of attachment of lipoic acid to apoenzymes pyruvate dehydrogenase (PDHc), α-ketoglutarate dehydrogenase (α-KGDHc) and branched chain α-keto acid dehydrogenase (BCKDHc) complexes.

Methods

Exome capture was performed in a boy who developed Leigh disease following a gastroenteritis and had combined PDH and α-KGDH deficiency with a unique amino acid profile that partly ressembled E3 subunit (dihydrolipoamide dehydrogenase / DLD) deficiency. Functional studies on patient fibroblasts were performed. Lipoic acid administration was tested on the LIPT1 ortholog lip3 deletion strain yeast and on patient fibroblasts.

Results

Exome sequencing identified two heterozygous mutations (c.875C?>?G and c.535A?>?G) in the LIPT1 gene that encodes a mitochondrial lipoyltransferase which is thought to catalyze the attachment of lipoic acid on PDHc, α-KGDHc, and BCKDHc. Anti-lipoic acid antibodies revealed absent expression of PDH E2, BCKDH E2 and α-KGDH E2 subunits. Accordingly, the production of ¹⁴CO₂ by patient fibroblasts after incubation with ^14Cglucose, ^14Cbutyrate or ^14C3OHbutyrate was very low compared to controls. cDNA transfection experiments on patient fibroblasts rescued PDH and α-KGDH activities and normalized the levels of pyruvate and 3OHbutyrate in cell supernatants. The yeast lip3 deletion strain showed improved growth on ethanol medium after lipoic acid supplementation and incubation of the patient fibroblasts with lipoic acid decreased lactate level in cell supernatants.

Conclusion

We report here a putative case of impaired free or H protein-derived lipoic acid attachment due to LIPT1 mutations as a cause of PDH and α-KGDH deficiencies. Our study calls for renewed efforts to understand the mechanisms of pathology of lipoic acid-related defects and their heterogeneous biochemical expression, in order to devise efficient diagnostic procedures and possible therapies.

     

Targeting a heterologous protein to multiple plant organelles via rationally designed 5′ mRNA tags

Background

Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments.

Results

We designed novel hybrid signal sequences for multiple-compartment localization and characterize their function when fused to GFP in Nicotiana benthamiana leaf tissue. TriTag-1 and TriTag-2 use alternative splicing to generate differentially localized GFP isoforms, localizing it to the chloroplasts, peroxisomes and cytosol. TriTag-1 shows a bias for targeting the chloroplast envelope while TriTag-2 preferentially targets the peroxisomes. TriTag-3 embeds a conserved peroxisomal targeting signal within a chloroplast transit peptide, directing GFP to the chloroplasts and peroxisomes.

Conclusions

Our novel signal sequences can reduce the number of cloning steps and the amount of genetic material required to target a heterologous protein to multiple locations in plant cells. This work harnesses alternative splicing and signal embedding for engineering plants to express multi-functional proteins from single genetic constructs.

     

The incidentally diagnosed adult congenital heart disease during routine medical health checkups in 27,897 Koreans at a single center over seven years

Background

The rate of incidentally diagnosed congenital heart disease (CHD) in adulthood has not been reported. The aim of this study was to investigate the detection rate of CHD in adults by routine, general health checkups.

Methods

Data was acquired from 222,401 patients older than 19?years who participated in general health checkups from January 2010 to December 2016. We excluded persons who did not undergo echocardiography during the general health checkups, who underwent echocardiography prior to the health checkups, and who were previously diagnosed with CHD.

Results

Among the 27,897 patients, who were included in the final analysis, 293 cases were newly diagnosed as CHD, and the overall detection rate was 1.05%. The mean age of patients with CHD was 48.7?±?21.5?years, and most of them were female (n?=?187, 63.8%). More than two-thirds were between the third and fifth decade of life, and only six patients (2.04%) were older than 70?years. The most common type was bicuspid aortic valve (n?=?155). Interestingly, Ebstein’s anomaly that required surgical repair was detected in five persons.

Conclusions

During general health checkup, there were cases of severe CHD that required cardiac surgery upon diagnosis.

     

MiR-9 enhances the sensitivity of A549 cells to cisplatin by inhibiting autophagy

Objectives

To demonstrate that miR-9 inhibits autophagy by down-regulating Beclin1 and thus enhances the sensitivity of A549 cells to cisplatin.

Results

MiR-9 inhibited Beclin1 expression by binding to its 3′UTR. The inhibition decreased the cisplatin-induced autophagy in A549 cells, evidenced by the decreased expression of LC3II and GFP-LC3 puncta and the increased expression of P62. Upregulation of miR-9 level enhanced the sensibility of A549 cells to cisplatin and increased the cisplatin-induced apoptosis. Overexpression of Beclin1 reversed above effects of miR-9 mimics, cisplatin-induced autophagy was increased and apoptosis was decreased.

Conclusions

MiR-9 inhibits autophagy via targeting Beclin1 3′UTR and thus enhances cisplatin sensitivity in A549 cells.