Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines

We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54 kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2 days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism.     

Heat shock protein 90 regulates the stability of MEKK3 in HEK293 cells

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the conformational maturation and function of certain signaling proteins. Hsp90 inhibitors cause the inactivation, destabilization and eventual degradation of Hsp90 client proteins through occupying the ATP/ADP binding pocket of Hsp90. In the present study, we found that Hsp90 interacted with MEKK3 in HEK293 cells. Hsp90 inhibitors reduced the level of endogenous MEKK3 in time- and dose-dependent manners, and this decrease was reversed by Hsp90 overexpression. In addition, Hsp90 RNAi destabilized MEKK3. A selective inhibitor of Hsp90, geldanamycin (GA), shortened MEKK3 half-life, and induced ubiquitination and proteasomal degradation of MEKK3. These results strongly suggested that Hsp90 could work as the molecular chaperone of MEKK3.     

钙离子对293细胞结团和生长的影响

分别在有血清和无血清条件下、方瓶和转瓶中考察了Ca²⁺ 对2 93细胞结团和生长的影响。通过实验发现,Ca²⁺ 浓度在0 1～1 0mmol L范围内对2 93细胞的贴壁和结团性质有显著影响,而对生长影响不大。结果表明:有血清贴壁培养时,较高的Ca²⁺ 浓度有利于细胞贴壁;无血清悬浮培养中,Ca²⁺ 浓度越高,细胞结团越严重,细胞结团达到平衡后的平均粒径(D ,μm)与Ca²⁺ 浓度(c,mmol L)在0.1～0.5mmol L范围内可用一次函数D =58.65c +16.96描述,细胞结团尺寸是可调控的;而细胞在不同的Ca²⁺ 浓度下有相似的生长规律。     

High-level expression and purification of the human bradykinin B(2) receptor in a tetracycline-inducible stable HEK293S cell line

The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.     

Orbital shaker technology for the cultivation of mammalian cells in suspension

For large-scale applications in biotechnology, cultivation of mammalian cells in suspension is an essential prerequisite. Typically, suspension cultures are grown in glass spinner flasks filled to less than 50% of the nominal volume. We propose a superior system for suspension cultures of mammalian cells based on orbital shaker technology. We found that "square-shaped" bottles (square bottles) provide an inexpensive but efficient means to grow HEK-293 EBNA and CHO-DG44 cells to high density. Cultures in agitated 1-L square bottles exceeded the performance of cultures in spinner flasks, reaching densities up to 7 x 10(6) cells/mL for HEK-293 EBNA cells and 5 x 10(6) cells/mL for CHO-DG44 cells in comparison to (2.5-4) x 10(6) cells/mL for cultures of the same cells grown in spinner flasks. For 1-L square bottles, optimal cell growth and viability were observed with a filling volume of 30-40% of the nominal volume and an agitation speed of 130 rpm at a rotational diameter of 2.5 cm. Transient reporter gene expression following gene delivery by calcium phosphate-DNA co-precipitation was the same or slightly better for HEK-293 EBNA cells grown in square bottles as compared to spinner flasks. Reductions in cost, simplified handling, and better performance in cell growth and viability make the agitated square bottle a new and very promising tool for the cultivation of mammalian cells in suspension.     

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells

Serotonin 5-HT4 receptor isoforms are G protein-coupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT4R ligands did not influence the constitutive energy transfer of the h5-HT4(d)R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT4(d)R and h5-HT4(g)R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT4R and beta2-adrenergic receptor (beta2AR) led to their heterodimerization. Given the large number of h5-HT4R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.     

KCNE3 truncation mutants reveal a bipartite modulation of KCNQ1 K+ channels

The five KCNE genes encode a family of type I transmembrane peptides that assemble with KCNQ1 and other voltage-gated K(+) channels, resulting in potassium conducting complexes with varied channel-gating properties. It has been recently proposed that a triplet of amino acids within the transmembrane domain of KCNE1 and KCNE3 confers modulation specificity to the peptide, since swapping of these three residues essentially converts the recipient KCNE into the donor (Melman, Y.F., A. Domenech, S. de la Luna, and T.V. McDonald. 2001. J. Biol. Chem. 276:6439-6444). However, these results are in stark contrast with earlier KCNE1 deletion studies, which demonstrated that a COOH-terminal region, highly conserved between KCNE1 and KCNE3, was responsible for KCNE1 modulation of KCNQ1 (Tapper, A.R., and A.L. George. 2000 J. Gen. Physiol. 116:379-389.). To ascertain whether KCNE3 peptides behave similarly to KCNE1, we examined a panel of NH(2)- and COOH-terminal KCNE3 truncation mutants to directly determine the regions required for assembly with and modulation of KCNQ1 channels. Truncations lacking the majority of their NH(2) terminus, COOH terminus, or mutants harboring both truncations gave rise to KCNQ1 channel complexes with basal activation, a hallmark of KCNE3 modulation. These results demonstrate that the KCNE3 transmembrane domain is sufficient for assembly with and modulation of KCNQ1 channels and suggests a bipartite model for KCNQ1 modulation by KCNE1 and KCNE3 subunits. In this model, the KCNE3 transmembrane domain is active in modulation and overrides the COOH terminus' contribution, whereas the KCNE1 transmembrane domain is passive and reveals COOH-terminal modulation of KCNQ1 channels. We furthermore test the validity of this model by using the active KCNE3 transmembrane domain to functionally rescue a nonconducting, yet assembly and trafficking competent, long QT mutation located in the conserved COOH-terminal region of KCNE1.