我们同时推荐您以High Five和Sf9这两类细胞来小规模尝试蛋白转染生产实验。通常每毫升的Sf9细胞培养物所生成的蛋白产物稍多,因为它们可于3–4 x 10E6个细胞/mL的密度下完成转染。High Five在大于2 x 10E6个细胞/mL的密度下就已完全进入对数生长期,因此它们不宜在高于2 x 10E6个细胞/mL的密度下进行转染。
昆虫细胞远比许多哺乳动物细胞系更为脆弱。这些细胞在过度生长和过度分离之后,会比哺乳动物细胞出现更多损伤。在悬浮培养时一定不要让细胞密度超过8 x 10E6细胞/mL或以低于0.5 x 10E6个细胞/mL的密度下生长。昆虫细胞需要略高于哺乳动物细胞的渗透压条件(340 μOsM)。昆虫细胞需要大量的O2,特别是在蛋白表达阶段。昆虫细胞培养基比哺乳动物培养基要酸得多(pH6.0-6.4)。昆虫细胞培养基是基于磷酸缓冲体系的。因此,无需使用CO2来维持pH值。
'OX40 is a member of the tumor necrosis factor (TNF) receptor superfamily and known to be an important costimulatory molecule expressed on activated T cells. To investigate the role of costimulation of OX40 in human immunodeficiency virus type 1 (HIV-1) infection by its natural ligand, gp34, the OX40-transfected ACH-2 cell ... More
Kinesin Superfamily Motor Protein KIF17 and mLin-10 in NMDA Receptor-Containing Vesicle Transport
'Experiments with vesicles containing N-methyl-D-aspartate (NMDA)receptor 2B (NR2B subunit) show that they are transported alongmicrotubules by KIF17, a neuron-specific molecular motor in neuronaldendrites. Selective transport is accomplished by direct interaction of theKIF17 tail with a PDZ domain of mLin-10 (Mint1/X11), which is aconstituent of a large protein complex including mLin-2 ... More
Alphaherpesvirus origin-binding protein homolog encoded by human herpesvirus 6B, a betaherpesvirus, binds to nucleotide sequences that are similar to ori regions of alphaherpesviruses.
Authors:Inoue N, Dambaugh TR, Rapp JC, Pellett PE
Journal:J Virol
PubMed ID:8207791
'We previously identified a human herpesvirus 6B (HHV-6B) homolog of the alphaherpesvirus origin-binding protein (OBP), exemplified by the herpes simplex virus type 1 UL9 gene product. This finding is of particular interest because HHV-6B is otherwise more closely related to members of the betaherpesvirus subfamily. The prototypic betaherpesvirus, human cytomegalovirus, ... More
Interaction of human nuclear topoisomerase I with guanosine quartet-forming and guanosine-rich single-stranded DNA and RNA oligonucleotides.
'Human nuclear DNA topoisomerase I (top1) plays a crucial role in DNA replication, transcription, and chromosome condensation. In this study, we show that intra- and intermolecular guanosine quartets (G-quartets) can inhibit top1-mediated DNA cleavage at a high affinity site. Top1-mediated DNA cleavage was also inhibited by a 16-mer single-stranded oligodeoxynucleotide ... More
Purification and characterization of human DNA damage checkpoint Rad complexes.
Authors: Lindsey-Boltz L A; Bermudez V P; Hurwitz J; Sancar A;
Journal:Proc Natl Acad Sci U S A
PubMed ID:11572977
'Checkpoint Rad proteins function early in the DNA damage checkpoint signaling cascade to arrest cell cycle progression in response to DNA damage. This checkpoint ensures the transmission of an intact genetic complement to daughter cells. To learn about the damage sensor function of the human checkpoint Rad proteins, we purified ... More