Selection Of High-producing Cell Lines Research Articles

Selecting cells expressing high levels of recombinant proteins using the GPI-anchored protein with selenocysteine system

Most biopharmaceutical proteins are produced in mammalian cells because they have the advantageous capacity for protein folding, assembly, and posttranslational modifications. To satisfy the increasing demand for these proteins for clinical purposes and studies, traditional methods to improve protein productivity have included gene amplification, host cell engineering, medium optimization, and screening methods. However, screening and selection of high-producing cell lines remain complex and time consuming. In this study, we established a glycosylphosphatidylinositol (GPI)-anchored protein with a selenocysteine (GPS) system to select cells producing high levels of target secretory proteins. Recombinant lysosomal acid lipase (LIPA) and α-galactosidase A (GALA) were fused with a GPI attachment signal sequence and a selenocysteine insertion sequence after an in-frame UGA codon. Under these conditions, most of the recombinant proteins were secreted into the culture medium, but some were found to be GPI-anchored proteins on the cell surface. When sodium selenite was supplied into the culture medium, the amount of GPI-anchored LIPA and GALA was increased. High-expressing cells were selected by detecting surface GPI-anchored LIPA. The GPI-anchored protein was then eliminated by knocking out the GPI biosynthesis gene PIGK, in these cells, all LIPA was in secreted form. Our system provides a promising method of isolating cells that highly express recombinant proteins from large cell populations.

Bioactive secondary metabolites produced in the author's laboratory by tissue culture techniques

SUMMARY Plant tissue culture techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant. Single cells, plant cells without cell walls (protoplasts), pieces of leaves, stems or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones. Cultured plant cells can be offered alternative sources over whole plant cultivation for the production of secondary metabolites. The explants, such as plant leaves, stems, roots, meristems etc. were used for cultivation and establishment of plant cell and tissue cultures. Unfortunately, most often plant cell cultures technologies not lead to an enhancement in secondary metabolites production. Therefore, Strain improvement, methods for the selection of high-producing cell lines, and medium optimizations must be considered to improve the production of secondary metabolites. Several types of cell culture methods are being used to produce bioactive compounds. Cell suspension cultures are preferred for production large amounts of cells for quantitative or qualitative analytical studies. This review refers to certain bioactive secondary metabolites produced in the author’s laboratory by different tissue cultures techniques.

A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures.

Plant cell and tissue cultures can be established routinely under sterile conditions from explants, such as plant leaves, stems, roots, meristems etc for both the ways for multiplication and extraction of secondary metabolites. Strain improvement, methods for the selection of high-producing cell lines, and medium optimizations can lead to an enhancement in secondary metabolite production. However, most often trials with plant cell cultures fail to produce the desired products. In such cases, strategies to improve the production of secondary metabolites must be considered. One of the main problems encountered is the lack of basic knowledge of the biosynthetic routes, and mechanisms responsible for the production of plant metabolites. Where the productivity of the desired metabolites is limited by the lack of particular precursors, biotransformation using an exogenous supply of biosynthetic precursors, genetic manipulation and metabolic engineering may improve the accumulation of compounds. Feedback inhibition of metabolic enzymes as well as inhibition of membrane transport can be eliminated by the accumulation of synthesized products in a second phase introduced into the aqueous medium. Organ cultures and in vitro biomass production often have sites of synthesis and storage of secondary metabolites in separate compartments. Elicitors, compounds triggering the formation of secondary metabolites, can be abiotic or biotic. Natural elicitors include polysaccharides such as pectin and chitosan, which are also used in the immobilization and permeabilization of plant cells. Immobilization with suitable bioreactor system provides several advantages, such as continuous process operation, but for the development of an immobilized plant cell culture process, natural or artifically induced secretion of the accumulated product into the surrounding medium is necessary. The present review highlights the nature, applications, perspective and scale up methods for the production of valuable secondary metabolites in vitro.   Key words: Plant cell cultures, secondary metabolites, biotransformation, biosynthetic pathways, elicitation, immobilization, bioreactors.