Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector.

Abstract

A cDNA coding for human interleukin 2 (IL-2) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus produced high levels of Mr 15,500 IL-2 polypeptide, the majority of which was secreted into the culture medium during infection. The recombinant IL-2 was able to stimulate the growth of an IL-2-dependent cell line. The N-terminal amino acid sequence of the insect-derived IL-2 was identical to that of natural IL-2. Thus, a mammalian signal peptide was recognized and properly removed in insect cells.

Baculovirus Expression Vectors: the Requirements for High Level Expression of Proteins, Including Glycoproteins

“…The pAcRP6 vector lacks only 7 bp of the 5' upstream sequences (in addition to those of the coding region). Smith et al (1985) had previously reported high levels of expression of interleukin 2 (IL-2) when using a vector (pAc373) that lacked the same nucleotides of the leader sequence as pAcRP6. In order to determine the effect of the integrity of the leader sequence on the expression of the HA gene several new transfer vectors were made with variable lengths of leader sequence (Fig.…”

Expression and purification of human IL-2 protein from Escherichia coli

“…After recombinant protein technology, IL-2 was expressed after isolation of IL-2 mRNA from human leukemic T-cell line, followed by cloning of the complementary cDNA into a suitable vector. The cloned IL-2 was expressed in different hosts such as E. coli (Ju et al, 1987;Devos et al, 1983;Rosenberg et al, 1984), insect cells (Smith et al, 1985), and monkey COS cells (Taniguchi et al, 1983). Most of the expressed IL-2 in E. coli was reported to be insoluble (forms IBs), however, the recombinant IL-2 was still retaining its biological activity even after extraction from SDS polyacrylamide gel (Devos et al, 1983).…”

Identification of Functionally Important Residues of the Silkmoth Pheromone Biosynthesis-activating Neuropeptide Receptor, an Insect Ortholog of the Vertebrate Neuromedin U Receptor

“…The elution profile began with an isocratic flow of 0% B for 2.5 min, followed by a linear gradient from 0 to 75% B in 15 , and the PBANR-EGFP mutants was assessed using a confocal scanning laser microscope. Sf9 insect cells, derived from the fall armyworm Spodoptera frugiperda (57,58), were adherently cultured on 35-mm glass bottom dishes (Asahi Glass, Tokyo, Japan) at 28°C for 2 days with 1000 l of IPL-41 (Invitrogen) supplemented with 10% FBS (Nichirei Bioscience, Tokyo, Japan). Cells were transfected with 5 g of each PBANR-EGFP expression plasmid using 10 l of Cellfectin II (Invitrogen) and incubated at 28°C for 16 h. Transfected cells were washed with 10% FBS/ IPL-41 containing 200 g/ml kanamycin and incubated at 28°C for 24 h. To examine co-localization of RR-C10PBAN R2K with the PBANR-EGFP mutants, cells were incubated in 500 l of IPL-41 containing 100 nM RR-C10PBAN R2K in the dark at 4°C for 60 min and then washed three times with PBS and fixed with 4% formaldehyde in PBS.…”