Expression of Human Recombinant CD23 in Insect Cells

Abstract

Human CD23 (low affinity receptor for IgE) has been expressed in insect cells (Sf9) using the baculovirus expression system and the baculovirus transfer vector pAc373. Insect cells infected with a recombinant baculovirus coding for CD23 synthesized a polypeptide not found in wild-type infected insect cells that had antigenic properties similar to natural CD23 produced in RPMI 8866 cells. Surface expression of recombinant CD23 was demonstrated by its ability to bind IgE. Recombinant CD23 expressed in insect cells had a slightly lower molecular weight (43 kDa) than that of natural CD23 (45 kDa) from RPMI 8866 cells as detected by SDS-PAGE followed by Western-blotting. Affinity-purified recombinant CD23 from infected insect cells showed B-cell growth promoting activitiy. These observations demonstrate for the first time that biologically active recombinant CD23 can be produced by the baculovirus expression system, thus providing a useful source of recombinant material to elucidate the biological functions of CD23.

Recombinant 25‐kDa CD23 and interleukin 1α promote the survival of germinal center B cells: evidence for bifurcation in the development of centrocytes rescued from apoptosis

“…Thus, within germinal centers, CD23 on FDC could serve a dual function: (a) as a membrane molecule providing homing, adhesion and, possibly, growth signals during the initial B blast reaction [3,7]; (b) as a soluble factor driving the further differentiation of rescued centrocytes. With regard to the former, we have recently reported that isolated, full-length CD23 (both natural and recombinant) is capable of providing a mitogenic signal to preactivated B lymphocytes [14,171.The latter possibility has been addressed in the present study.…”

“…The transfer vector pAcUW2-IL 2-25 for the expression of 25-kDa CD23 was constructed as follows: pAc373-45 [14] was cleaved with BamHI and Sau3A and the 631-bp fragment coding for the 3' end of CD23 was isolated from low-melt agarose gels. A synthetic oligonucleotide pair To generate a source of recombinant 25-kDa CD23, insect cells (Sf9) were infected with clone H2 at a multiplicity of 1 and cell culture SN were harvested 3 days after infection.…”

Demonstration of a second ligand for the low affinity receptor for immunoglobulin E (CD23) using recombinant CD23 reconstituted into fluorescent liposomes.

“…RPMI 8866 has been reported to proliferate in response to recombinant CD23 (9), and germinal center B calls have been shown to respond to soluble CD23 with IL-1 by increased survival (10). Moreover, we show here that a subset of T calls also binds the CD23 liposomes.…”

“…The recombinant membrane form of CD23 was expressed in insect cells using the baculovirus expression system (9), and purified to homogeneity by immunoatiinity chromatography, resulting in a preparation that was 95% pure (Fig. 1 A) as assessed by coomassie blue-stained SDS-PAGE and Western blotting analysis (Fig.…”

“…An increasing number of reports have demonstrated that CD23 also plays a role in non-IgE-related activities such as B cell growth (8,9), germinal center B cell survival (10), prothymocyte maturation (11), myeloid precursor proliferation (12), and antigen presentation (13). This latter observation, together with the earlier finding that CD23 was spatially associated with the MHC class II molecule HLA-DR on the B cell surface (14), suggested that CD23 may be involved in T ceU-B cell interactions.…”