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Rademacher, Thomas (3)
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Year of Publication
The Induction of Recombinant Protein Bodies in Different Subcellular Compartments Reveals a Cryptic Plastid-Targeting Signal in the 27-kDa γ-Zein Sequence
Frontiers in Bioengineering and Biotechnology
Naturally occurring storage proteins such as zeins are used as fusion partners for recombinant proteins because they induce the formation of ectopic storage organelles known as protein bodies (PBs) where the proteins are stabilized by intermolecular interactions and the formation of disulfide bonds. Endogenous PBs are derived from the endoplasmic reticulum (ER). Here, we have used different targeting sequences to determine whether ectopic PBs composed of the N-terminal portion of mature 27 kDa γ-zein added to a fluorescent protein could be induced to form elsewhere in the cell. The addition of a transit peptide for targeting to plastids causes PB formation in the stroma, whereas in the absence of any added targeting sequence PBs were typically associated with the plastid envelope, revealing the presence of a cryptic plastid-targeting signal within the γ-zein cysteine-rich domain. The subcellular localization of the PBs influences their morphology and the solubility of the stored recombinant fusion protein. Our results indicate that the biogenesis and budding of PBs does not require ER-specific factors and therefore, confirm that γ-zein is a versatile fusion partner for recombinant proteins offering unique opportunities for the accumulation and bioencapsulation of recombinant proteins in different subcellular compartments.
protein bodies; molecular farming; subcellular targeting; intermembrane space; plastid import; recombinant protein
Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells
Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10).
We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5′-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host.
We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression.
The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation: Down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant-based expression platforms
Torres Acosta, Juan Antonio
The tobacco-related species Nicotiana benthamiana has recently emerged as a promising host for the manufacturing of protein therapeutics. However, the production of recombinant proteins in N. benthamiana is frequently hampered by undesired proteolysis. Here, we show that the expression of the human anti-HIV antibodies 2F5, 2G12, and PG9 in N. benthamiana leaves leads to the accumulation of discrete heavy chain-derived degradation products of 30–40 kDa. Incubation of purified 2F5 with N. benthamiana intercellular fluid resulted in rapid conversion into the 40-kDa fragment, whereas 2G12 proved largely resistant to degradation. Such a differential susceptibility to proteolytic attack was also observed when these two antibodies were exposed to various types of proteinases in vitro. While serine and cysteine proteinases are both capable of generating the 40-kDa 2F5 fragment, the 30-kDa polypeptide is most readily obtained by treatment with the latter class of enzymes. The principal cleavage sites reside within the antigen-binding domain, the VH–CH1 linker segment and the hinge region of the antibodies. Collectively, these results indicate that down-regulation of endogenous serine and cysteine proteinase activities could be used to improve the performance of plant-based expression platforms destined for the production of biopharmaceuticals.
Antibodies; Biopharmaceutical; Nicotiana; Proteinase; Protein stability
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