Elsevier

Enzyme and Microbial Technology

Volume 33, Issue 4, 10 September 2003, Pages 466-471
Enzyme and Microbial Technology

Purification of recombinant 30K protein produced in Escherichia coli and its anti-apoptotic effect in mammalian and insect cell systems

https://doi.org/10.1016/S0141-0229(03)00149-2Get rights and content

Abstract

A gene coding for an anti-apoptotic 30K protein originating from the silkworm, Bombyx mori, was cloned into the vector pET 22b(+) to produce this protein in Escherichia coli. The 30K gene was fused to a C-terminal His-tag to facilitate the separation process. The 30K protein was expressed in the form of an inclusion body. This was denatured, purified by single-step immobilized metal ion affinity chromatography (IMAC), and refolded by two different methods, i.e. on-column refolding and refolding by dilution after column chromatography. The latter method resulted in the higher separation yield of the 30K protein. The purified 30K protein effectively inhibited insect cell (Sf9) and mammalian cell (HeLa) apoptosis by addition to culture medium.

Introduction

Cell death is categorized as either apoptotic or necrotic. Apoptosis is a physiological cell death, which is morphologically distinguishable from necrosis [1]. Necrotic cells are characterized by an overall increase in size, mild clumping of chromatin and cell lysis, whereas apoptosis is accompanied by the condensation of nuclei and cytoplasm, the loss of microvilli, convolution of the plasma membrane, and nuclear and cell segmentation. Apoptosis occurs sporadically in all tissues throughout life and is a normal everyday occurrence; however, disproportionate apoptosis, either excessive or deficient may cause serious diseases.

A silkworm, Bombyx mori, has been used effectively in various biological researches [2]. The production of recombinant protein in an insect cell baculovirus system was increased by supplementing the medium with silkworm hemolymph (SH) [3]. SH increases baculovirus-infected insect cell longevity [4], [5], [6]. Moreover, it has been shown that SH inhibits apoptosis in insect, mammalian, and human cell systems [7], [8] indicating that SH contains a component that inhibits apoptosis. More recently, this anti-apoptotic component was isolated from SH and characterized [9].

The fraction of SH with the highest activity was found to contain 30K proteins, which are a specific type of plasma protein called ‘storage proteins’. These proteins constitute a group of structurally related proteins of approximate molecular weight 30,000 Da [10], [11]. In this study, the 30K protein encoded by the 30Kc6 gene of B. mori was expressed in Escherichia coli and purified for the production of recombinant anti-apoptotic protein.

Section snippets

Bacterial strain and plasmid

E. coli BL21(DE3) was used as the host for gene expression. A plasmid containing the 30K protein cDNA (30Kc6, GenBank accession number: X54735) was kindly provided by S. Izumi (Department of Biology, Tokyo Metropolitan University). The 30K protein cDNA was amplified by PCR and the amplified PCR products were cloned into E. coli expression vector, pET 22b(+). During this step a signal sequence contained in 30Kc6 was removed, and the vector was designed to express the 30K protein with a 6× His

Results and discussion

The expression of 30K protein was induced using 1 mM IPTG. The time course of its expression is shown in Fig. 1. The number in each lane represents time (hours) post-induction. The 30K protein, which has the molecular weight of approximately 30,000 Da, was detected in the insoluble fraction of the cell lysate as shown in Fig. 1A, but not detected in the soluble fraction (Fig. 1B). The concentration of the 30K protein reached 0.5 g/l 20 h after IPTG induction. The molecular weight of the expressed

Acknowledgements

The authors wish to acknowledge the financial support provided by the Korea Science & Engineering Foundation through the Nano Bio-Electronic & System Center, Seoul National University, Seoul, South Korea.

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    During metamorphosis from larva to pupa, these proteins are transferred from the hemolymph to fat body cells and are deposited there until use [26,27]. We have demonstrated previously that silkworm hemolymph and 30 K proteins exhibit an anti-apoptotic effect in various cells by adding the protein to culture medium or by gene expression [18,28–39]. 30 K proteins also enhance production of recombinant EPO, interferon-β, and monoclonal antibodies; increase glycosylation, cell growth, and viability in various cells; and have an enzyme-stabilizing effect [17,40–46].

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