We have developed a versatile Escherichia coli expression system based on the use of E. coli thioredoxin (trxA) as a gene fusion partner. The broad utility of the system is illustrated by the production of a variety of mammalian cytokines and growth factors as thioredoxin fusion proteins. Although many of these cytokines previously have been produced in E. coli as insoluble aggregates or "inclusion bodies", we show here that as thioredoxin fusions they can be made in soluble forms that are biologically active. In general we find that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels. Two additional properties of E. coli thioredoxin, its ability to be specifically released from the E. coli cytoplasm by osmotic shock or freeze/thaw treatments and its intrinsic thermal stability, are retained by some fusions and provide convenient purification steps. We also find that the active-site loop of E. coli thioredoxin can be used as a general site for small peptide insertions, allowing for the high level production of soluble peptides in the E. coli cytoplasm.
The initial interaction between B cells and follicular dendritic cells (FDCs) appears to be essential for germinal center (GC) formation. To identify molecules regulating this interaction, we generated FDC-staining monoclonal antibodies (mAbs) and screened them for their ability to block FDC-mediated costimulation of growth and differentiation of CD40-stimulated B cells. Using one of the inhibitory mAbs, 8D6, we expression cloned the cDNA encoding the 8D6 antigen (Ag) from a human FDC line, HK. The 8D6 Ag is a novel protein of 282 amino acids that is expressed abundantly on FDCs. Monolayers of COS cells transiently transfected with the 8D6 Ag cDNA stimulate B cell growth. The mAb 8D6 blocks the costimulatory function completely. The inhibitory activity of the mAb 8D6 was demonstrated to be due to an inhibition of cell cycle progression of CD40 ligand–stimulated GC B cells. In addition, the mAb 8D6 inhibits the growth of a lymphoma of GC origin, L3055, which depends on FDCs or HK cells for its growth. These findings suggest that the primary function of FDCs in the GC is to stimulate B cell growth. An FDC signal molecule, 8D6 Ag, may be an important molecule to mediate this function.
Flt4 is a receptor protein tyrosine kinase that is expressed in the adult lymphatic endothelium and high endothelial venules. We have used a BIAcore assay to identify rodent and human cell conditioned media containing the ligand of Flt4 (Flt4-L). Receptor-based anity chromatography was used to purify this growth factor, followed by amino acid sequencing and molecular cloning of the murine cDNA, the orthologue of human vascular endothelial growth factor-C and vascular endothelial growth factor related protein. The murinē t4-L gene was localized to chromosome 8 and demonstrated to be widely expressed. Flt4-L was found to have a hydrophobic signal sequence and a pro-peptidelike sequence that is removed to generate the mature Nterminus. In addition, the C-terminal region of Flt4-L has four repeats of a cysteine-rich motif that is presumably also proteolytically processed to generate the 21 000 M r polypeptide subunit of the Flt4-L homodimer. Recombinant Flt4-L activated Flt4 as judged by induction of tyrosyl phosphorylation, and induced mitogenesis in vitro of lymphatic endothelial cells.Keywords: Flt4; Flt4 ligand; VEGF-C; BIAcoreTo identify a source of the Flt4 ligand, we used a Flt4-Fc fusion protein. This consists of the extracellular region of murine Flt4 (Finnerty et al., 1993) fused to the hinge-C H 2-C H 3 domains of human immunoglobulin g1. The conditioned media (CM) of over 100 dierent cell lines were screened for binding to Flt4-Fc using a BIAcore 2000 instrument. The media conditioned by the murine embryonic liver cell line, BNL 1NG A.2, human bladder carcinoma 5637, and bualo rat BRL 3A each showed binding to Flt4-Fc, but not human IgG1 (Figure 1). The binding was speci®c because when the CM was coinjected with excess Flt4-Fc, but not human IgG1 or KIT-Fc, the signal was inhibited (Figure 1). The presence of Flt4 ligand (Flt4-L) in BNL 1NG A.2 CM was con®rmed by demonstrating that it activated the Flt4 receptor protein tyrosine kinase. CHO cells stably transfected with a murine Flt4 cDNA expression vector were treated with 50-fold concentrated BNL 1NG A.2 CM for 10 min at 378C. This resulted in a marked increase in tyrosyl phosphorylation of Flt4 (Figure 2a, lane 1) relative to mock stimulated samples (Figure 2a, lane 2).Six micrograms of Flt4-L were puri®ed from 15L of BNL 1NG A.2 CM using Flt4-Fc anity chromatography. Flt4-Fc (100 mg/ml) or KIT-Fc (200 mg/ml) were directly immobilized to 1 mL Pharmacia HiTrap NHS columns. A volume of 50 ml of concentrated BNL 1NG A.2 CM was loaded ®rst onto the KIT-Fc column to adsorb nonspeci®c binding, then onto the Flt4-Fc column. The columns were washed and separately eluted with 24 mM hydrochloric acid. As expected, only fractions from the Flt4-Fc anity column had binding to Flt4-Fc on the BIAcore assay. The speci®c activity in response units (RU)/mg was determined using RU that were in the linear range of RU versus concentration of Flt4-L puri®cation fractions. The speci®c activity of Flt4-L from BNL 1NG A.2 was 4.2610 6 RU/mg, an increase of 98 455-fold after t...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.