A new transient expression system for large-scale production of recombinant proteins in plants based on air-brushing an Agrobacterium suspension

During the last two decades, the production of pharmaceutical proteins in plants evolved from proof of concept to established technology adopted by several biotechnological companies. This progress is particularly based on intensive research starting stable genetic transformation and moving to transient expression. Due to its advantages in yield and speed of protein production transient expression platforms became the leading plant-based manufacturing technology. Current transient expression methods rely on Agrobacteriummediated delivery of expression vectors into plant cells. In recent years, great advances have been made in the improvement of expression vectors, host cell engineering as well as in the development of commercial manufacturing processes. Several GMP-certified large-scale production facilities exist around the world to utilize agroinfiltration method. A number of pharmaceutical proteins produced by transient expression are currently in clinical development. The great potential of transient expression platform in respect to rapid response to emerging pandemics was demonstrated by the production of experimental ZMapp antibodies against Ebola virus as well as influenza vaccines. This review is focused on current design, status and future perspectives of plant transient expression system for the production of biopharmaceutical proteins.

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cDNA cloning and heterologous expression of a wheat proteinase inhibitor of subtilisin and chymotrypsin (WSCI) that interferes with digestive enzymes of insect pests

Biological Chemistry ◽

10.1515/bc.2005.046 ◽

2005 ◽

Vol 386 (4) ◽

pp. 383-389 ◽

Cited By ~ 11

Author(s):

Simone Di Gennaro ◽

Anna G. Ficca ◽

Daniela Panichi ◽

Elia Poerio

Keyword(s):

Proteinase Inhibitor ◽

Large Scale ◽

Insect Pests ◽

Expression System ◽

Physiological Role ◽

Scale Production ◽

Insect Larvae ◽

E Coli ◽

Large Scale Production ◽

Degenerate Oligonucleotide

Abstract A cDNA encoding the proteinase inhibitor WSCI (wheat subtilisin/chymotrypsin inhibitor) was isolated by RT-PCR. Degenerate oligonucleotide primers were designed based on the amino acid sequence of WSCI and on the nucleotide sequence of the two homologous inhibitors (CI-2A and CI-2B) isolated from barley. For large-scale production, wsci cDNA was cloned into the E. coli vector pGEX-2T. The fusion protein GST-WSCI was efficiently produced in the bacterial expression system and, as the native inhibitor, was capable of inhibiting bacterial subtilisin, mammalian chymotrypsins and chymotrypsin-like activities present in crude extracts of a number of insect larvae (Helicoverpa armigera, Plodia interpunctella and Tenebrio molitor). The recombinant protein produced was also able to interfere with chymotrypsin-like activity isolated from immature wheat caryopses. These findings support a physiological role for this inhibitor during grain maturation.

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Secretory Expression of YY(3-36) Peptide in E. coli

10.31219/osf.io/rn586 ◽

2019 ◽

Author(s):

Sorush Niknamian

Keyword(s):

Large Scale ◽

Peptide Yy ◽

Signal Sequence ◽

Expression System ◽

Complete Sequence ◽

Scale Production ◽

E Coli ◽

Large Scale Production ◽

Human Experiments ◽

Clinical Experiments

Obesity is the prime suspect in a wide frequency of diabetes type II and cardiovascular diseases worldwide. Recombinant YY (tyrosine-tyrosine) peptide is a locally acting hormone, controlling secretion in the digestive tract. Interestingly, it was later shown that a truncated version of YY peptide, YY(3-36) peptide, has the potential as an important biopharmaceutical in a fight against obesity. This peptide has shown promising results in human clinical experiments in appetite reduction in human experiments. To develop an economical expression system for large-scale production of the peptide in gram-negative bacteria, we have developed a chimeric gene for extracellular expression of this peptide with the assistance of signal sequence of asparaginase II from Escherichia coli. This system has the advantage of producing the complete sequence of YY(3-36) without any extra tags that require further removal with the assistance of expensive specific proteases and reduce the downstream steps significantly. Our results pave the way for the recombinant production of YY(3-36) peptide and further proves the efficacy of asparaginase II signal sequence as a communicator of foreign peptides and proteins into extracellular space of E. coli.

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Production of Recombinant Melittin by Auto-Induction in Escherichia coli

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.798-799.1007 ◽

2013 ◽

Vol 798-799 ◽

pp. 1007-1012

Author(s):

Wen He Zhu ◽

Wei Zhang ◽

Yan Li ◽

Jun Jie Xu ◽

Shi Jie Lv

Keyword(s):

Fusion Protein ◽

Large Scale ◽

Expression Vector ◽

Human Cancer ◽

Expression System ◽

Scale Production ◽

Recognition Sequence ◽

Large Scale Production ◽

N Terminus ◽

Gst Fusion Protein

Melittin is a novel peptide of biological activity isolated from bee venom. It has potential application value in medicine and agriculture. Here we encoded melittin gene with the EK recognition sequence in the N-terminus into expression vector pGEX-2T.The expressed fusion protein, which is about 29KDa, identified by Western Blot. To facilitate large-scale production of recombinant GST-fusion protein, we optimized different expression conditions to increase the overall production of the fusion protein. The production of the protein had increased about 10-fold when we used an auto-inducing medium. The GST fusion protein showed an equivalent activity with the natural melittin after digested by EK and can inhibited the proliferations of several human cancer lines. The expression system described in this study provides a feasible way for producing melittin in further studies.

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Plants as factories for production of biopharmaceutical and bioindustrial proteins: lessons from cell biologyThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

Canadian Journal of Botany ◽

10.1139/b06-069 ◽

2006 ◽

Vol 84 (4) ◽

pp. 679-694 ◽

Cited By ~ 22

Author(s):

Allison R. Kermode

Keyword(s):

Recombinant Proteins ◽

Protein Function ◽

Cell Biology ◽

Large Scale ◽

Production Systems ◽

Plant Cells ◽

Scale Production ◽

Functional Protein ◽

Large Scale Production ◽

Engineer Plant

Transgenic plants, seeds, and cultured plant cells are potentially one of the most economical systems for large-scale production of recombinant proteins for industrial and pharmaceutical uses. Biochemical, technical, and economic concerns with current production systems have generated enormous interest in developing plants as alternative production systems. However, various challenges must be met before plant systems can fully emerge as suitable, viable alternatives to current animal-based systems for large-scale production of biopharmaceuticals and other products. Aside from regulatory issues and developing efficient methods for downstream processing of recombinant proteins, there are at least two areas of challenge: (1) Can we engineer plant cells to accumulate recombinant proteins to sufficient levels? (2) Can we engineer plant cells to post-translationally modify recombinant proteins so that they are structurally and functionally similar to the native proteins? Attempts to improve the accumulation of a recombinant protein in plant cells require an appreciation of the processes of gene transcription, mRNA stability, processing, and export, and translation initiation and efficiency. Likewise, many post-translational factors must be considered, including protein stability, protein function and activity, and protein targeting. Moreover, we need to understand how the various processes leading from the gene to the functional protein are interdependent and functionally linked. Manipulation of the post-translational processing machinery of plant cells, especially that for N-linked glycosylation and glycan processing, is a challenging and exciting area. The functions of N-glycan heterogeneity and microheterogeneity, especially with respect to protein function, stability, and transport, are poorly understood and this represents an important area of cell biology.

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Large-Scale Production of a Soluble Human β-1,4-Galactosyltransferase Using a Saccharomyces cerevisiae Expression System

Protein Expression and Purification ◽

10.1006/prep.1995.1010 ◽

1995 ◽

Vol 6 (1) ◽

pp. 72-78 ◽

Cited By ~ 18

Author(s):

G.F. Herrmann ◽

C. Krezdorn ◽

M. Malissard ◽

R. Kleene ◽

H. Paschold ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Large Scale ◽

Expression System ◽

Scale Production ◽

Large Scale Production

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Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

Applied and Environmental Microbiology ◽

10.1128/aem.70.6.3292-3297.2004 ◽

2004 ◽

Vol 70 (6) ◽

pp. 3292-3297 ◽

Cited By ~ 28

Author(s):

Gerard M. Gibbs ◽

Barrie E. Davidson ◽

Alan J. Hillier

Keyword(s):

Fusion Protein ◽

Large Scale ◽

Expression System ◽

Reversed Phase ◽

Scale Production ◽

Gene Encoding ◽

Strong Activity ◽

E Coli ◽

Large Scale Production ◽

Bacterial Cell Membrane

ABSTRACT Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.

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