Jerdostatin, an RTS short disintegrin cloned from
Protobothrops jerdonii and recombinantly produced in
Escherichia coli, is a potent and specific antagonist of the α
1β
1 integrin. Jerdostatin ...selectively blocked the adhesion of α
1β
1-K562 cell to collagens I and IV
in vitro and angiogenesis
in vivo. Here we report the recombinant production of jerdostatin in a mammalian cell system, a prerequisite for developing a conditional transgenic mouse to investigate the effect of systemic expression of jerdostatin on tumor development. For proper export of jerdostatin, a secretion leader sequence was engineered at the protein’s N-terminus. A FLAG epitope was also included at the N-terminus of the mature disintegrin to facilitate its isolation and characterization of recombinant jerdostatin (rJerd). This pRc-CMV/FLAG-rJerd construct was transiently expressed in HEK-293 cells and was efficiently secreted into the culture medium. rJerd bound to recombinant soluble α
1β
1 integrin in a saturable and cation-independent manner. Soluble rJerd also inhibited the binding of α
1β
1 integrin to the CB3 fragment of collagen IV in a dose-dependent manner (IC
50 570 nM). Mammalian cell-expressed jerdostatin disrupted the adhesion of RuGli cells to collagen IV. Our results highlight pRc-CMV/FLAG-rJerd as a suitable construct for expressing soluble active α
1β
1-blocking jerdostatin in a mammalian cell system.
In response to sustained depolarization or prolonged bursts of activity in spiking cells, sodium channels enter long-lived non-conducting states from which recovery at hyperpolarized potentials ...occurs over hundreds of milliseconds to seconds. The molecular basis for this slow inactivation remains unknown, although many functional domains of the channel have been implicated. Expression studies in
Xenopus
oocytes and mammalian cell lines have suggested a role for the accessory β1 subunit in slow inactivation, but the effects have been variable. We examined the effects of the β1 subunit on slow inactivation of skeletal muscle (NaV1.4) sodium channels expressed in HEK cells. Co-expression of the β1 subunit impeded slow inactivation elicited by a 30-s depolarization, such that the voltage dependence was right shifted (depolarized) and recovery was hastened. Mutational studies showed this effect was dependent upon the extracellular Ig-like domain, but was independent of the intracellular C-terminal tail. Furthermore, the β1 effect on slow inactivation was shown to be independent of the negative coupling between fast and slow inactivation.
The expression of transgenes in mammalian cells is often at a low level mainly due to position effects from the neighboring chromatin context. To improve this, we have constructed a vector pCAM, ...which contains chloramphenicol acetyltransferase (CAT) reporter gene cassettes, driven by SV40 early promoter and flanked by two human β-globin MARs in
cis. We transfected this vector into the Chinese hamster ovary (CHO) cell line, and found that the level of CAT gene expression with MAR was effectively increased, about 5.493-fold higher than those without MARs. Moreover, the variations of CAT expression among individuals of transformants were decreased 2.670-fold. Our result also showed that MAR could increase the proportion of positive colonies in recombinants.
Previous work has shown that the MAR (matrix attachment region) could increase transgene expression in stably transfected CHO (Chinese‐hamster ovary) cells. To study the positional effect of MAR on ...transgene expression, three expression vectors were constructed which contained the human β‐globin MAR in different sites, including the vector with two MARs flanking the CAT (chloramphenicol acetyltransferase) expression cassette, one MAR at the 5′ or 3′ site. These vectors were transfected into CHO cells. The level of CAT gene expression was most effectively increased by two MARs flanking the CAT expression cassette. This increase was also seen when MAR was inserted at the 5′ site upstream of the expression cassette, whereas the transgene expression level decreased when MAR was inserted at the 3′ site downstream of the expression cassette. We have also shown that the transgene expression level is not directly proportional to the gene copy number, and gene copy number dependency does not exist.
The industrial production of recombinant proteins preferentially requires the generation of stable cell lines expressing proteins in a quick, relatively facile, and a reproducible manner. Different ...methods are used to insert exogenous DNA into the host cell, and choosing the appropriate producing cell is of paramount importance for the efficient production and quality of the recombinant protein. This review addresses the advances in recombinant protein production in mammalian cell lines, according to key patents from the last 30 years.
Purified protein expression level and quality are contingent upon specific host expression systems. This differential production is particularly observed for proteins of high molecular weight, ...hampering further structural studies. We developed an expression method aimed at producing proteins in Escherichia coli, insect, and mammalian systems. Our novel protocol was used to produce in large scale the full-length 160-kDa steroid receptor coactivator 1 (SRC-1), a coregulator of nuclear receptors. The results indicate that we can produce biologically active human SRC-1 in mammalian and insect cells in large scale.
As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become valuable reagents for immunotherapy and laboratory investigations. Additional applications for these ...reagents include flow cytometry, immunohistochemistry, and in vitro activity assays. To aid investigators in the generation of these reagents, the materials and methods required for producing Fc fusion proteins are described. The investigator's protein moiety of interest is genetically linked to the N-terminus of murine Fc and subsequently expressed in large quantity using a mammalian cell expression system. The resulting Fc fusion proteins are purified on a protein A column and may be stored for at least one year at -20 degrees C. The availability of easily purified, soluble Fc fusion proteins in such quantity can facilitate research in multiple fields of medicine and biotechnology.
Ion channels are integral membrane proteins that regulate membrane potentials and signaling of cells in response to various stimuli. The patch-clamp technique enables the study of single channels or ...a population of channels. The macroscopic recording approaches are powerful in revealing population-averaged behaviors of channels both under basal conditions and in response to various stimuli, modulators and drugs. On their own, however, these approaches can be insufficient for determinations of channel gating mechanisms as they do not accurately report channel open probabilities below 10(-2) to 10(-3). This obstacle can be overcome with the use of single-channel recording techniques. Single-channel recording techniques can be applied to one or a few channels to estimate P o over a larger range than macroscopic recordings. The combination of heterologous overexpression of ion channels with macroscopic and single-channel recordings can be applied to hundreds of channels to estimate P o between 1 and 10(-8). Here, we describe practical approaches of single-channel recordings that our laboratory utilizes. We also provide examples where the combined macroscopic and single channel approach can be employed to study gating mechanisms of the BK type, large conductance, Ca(2+) and voltage activated potassium channel in a mammalian expression system. The techniques presented should be generally applicable to the studies of ion channels in heterologous expression systems.
This work generated many truncated proteins and Glu
385 to Ala (E
385/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active ...endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E
385/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH
2-terminal sequence of L
33GRPSEEDEE. A species at 115 kDa and some other protein bands began with F
236VSSHRYV
243, indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R
235–F
236 peptide bond. This cleavage was not an autocatalytic process since the E
385/A mutants were also processed. Furthermore, a 52 kDa band with an NH
2-terminal sequence of L
800KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.
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