Development of Recombinant Protein Production System Using Mushroom

Abstract

The advent of recombinant protein production technology has led to a worldwide zeal for the development of protein pharmaceuticals in the past two decades. These protein pharmaceuticals or pharmaceutical candidates include functional regulators and supplements, enzyme activators and inhibitors, polyclonal and monoclonal antibodies, and various vaccines. In comparison with small chemical drugs, protein pharmaceuticals have high specificity and activity at relatively low concentrations. These features have made protein pharmaceuticals indispensable for combating human diseases. Human GH (hGH) has been available over 40 years for the treatment of children with GH deficiency. Human growth hormone (hGH) is mainly produced in the somatotrophic cells of the pituitary in brain and is the product of the GH-N gene. The most prominent effects of GH include promoting growth and having protein anabolic and lipolytic effects. As unlimited supplies of recombinant GH became available in 1985, it has been possible to investigate the metabolic effects of GH rather than just its well characterized growth-promoting effects. Recombinant human growth hormone is anabolic and enhances immune functions. Aside from its growth stimulation effects, hGH has been considered as a possible therapeutic agent for the treatment of the frailty associated with aging, osteoporosis, morbid obesity, cardiac failure, major thermal injury, and a variety of other acute and chronic catabolic conditions. IL-32, originally named NK cell transcript 4 (NK4), is the latest described inflammatory cytokine. Its transcripts are highly expressed in immune tissues. It is produced mainly by mitogen-activated lymphocytes, IFN-γ activated eCs, IL-12, IL-18 and IL-32-activated NK cells, and IL-18 gene transfected cells. IL-32 promotes a number of proinflammatory cytokines, such as TNF-α, IL-8, and MIP-2 in different cells. Human recombinant IL-32 induces the production of large amounts of several proinflammatory cytokines and chemokines, even in macrophage cell lines by activation of NF-κB and MAPK. The ability of IL-32 to potentiate inflammation is not the sole action of this cytokine. IL-32 stimulates prostaglandin (PG)E2 in human PBMCs, which are pivotal in inflammation. This indicates that IL-32 contributes to the mediation of cartilage and bone destruction in Rheumatoid Arthritis (RA). Therefore, IL-32 antibody may be effective to treat these diseases by virtue of its anti-inflammatory and probably analgesic properties. Among the edible mushrooms, the king oyster mushroom (Pleurotus eryngii) is one of the most popular mushrooms in Asia, Europe and North America. The increasing popularity of P. eryngii among consumers is due to its flavor, texture and shelf life. Commercial production of this species began in Italy in the mid 1970s and is produced in over a dozen other countries. Mushrooms have been used for the diet in many countries due to their good taste and nutritive value. Bioactive molecules have been isolated from mushrooms including lectin, nucleases, proteases, ribosome-inactivating proteins, ubiquitin-like protein, polysaccharides and polysaccharide-peptide and polysaccharide protein complexes. Agrobacterium tumefaciens mediated transformation of fungi was first reported in 1995. Further developments in Agrobacterium tumefaciens mediated transformation of fungi have taken place and several studies continued to optimize the technology. This study suggests a modified Agrobacterium-mediated method for the efficient transformation of hGH2 and hIL32 in Pleurotus eryngii. The binary vector, pCambia1304 was used for the initial transformation and transformants were selected by expression of selectable markers, such as hygromycin phosphotransferase gene (hygromycin resistance), β-glucuronidase (GUS) and Green Fluorescent Protein (GFP). Infiltrated samples transformed with pCambia1304 showed a wider GUS response than the co-cultivated in the 50㎍/㎖ hygromycin and cefotaxim selection medium. Transformants appeared at the margins of the tissue pieces after 9 to 14 days on selection medium. Particle bombardment offered another approach to improve Agrobacterium tumefaciens mediated transformation through the causing of micro wounds and the delivery of Agrobacterium deep into the target tissue. Also particle bombardment method had advantage to obtain transformants without use of antibiotics, cefotaxim. In particle bombardment, DNA was bound to 0.6㎛ gold particle on the tip of microcarriers. The macrocarrier is accelerated through a barrel towards the target tissue by hellum gas shock of 1,300 psi pressure. And then, the transformants were selected in the medium containing 50㎍/㎖ hygromycin. Transformants appeared at the margins of the tissue pieces after 7 to 10 days on selection medium. From both infiltration and particle bombardment methods, 13 putative transformants were screened. Transformants PCR analyses confirmed that the hGH2 and hIL32 gene was into the genome of Pleurotus eryngii. Expression of the transgenes was confirmed by GUS staining, Southern hybridization, Northern hybridization and Western blot analysis. Both transformation techniques described herein provide a practical method for using transgenic technology in the genetic improvement of this commercially important mushroom and represents an important tool for the molecular genetic analysis of biological processes in this species. Also, through the development of mushroom-contained recombinant protein health foods, functional foods and pharmaceutical raw materials, this study final aims to make growing contributions to life science, focusing on cures for disease and maintenance and promotion of good health.