Purification of recombinant aprotinin produced in transgenic corn seed: separation from CTI utilizing ion-exchange chromatography

Azzoni, Adriano Rodrigues; Takahashi, Koichi; Woodard, Susan L.; Miranda, Everson Alves; Nikolov, Z̆ivko L.

doi:10.1590/s0104-66322005000300001

Cited by 10 publications

(4 citation statements)

References 15 publications

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“…Overall, few detailed protocols for downstream processing have been published. Azzoni et al (2005) showed that replacing immobilized metal affinity chromatography (IMAC) with a cation exchange chromatography step achieved a tenfold improvement in the recovery and purity of recombinant aprotinin from transgenic maize. Similarly, Nandi et al (2005) showed that lactoferrin was efficiently extracted from rice flour in phosphate buffer (pH 7.0) containing up to 0.5 M NaCl.…”

Section: Downstream Processing and Purificationmentioning

confidence: 99%

Molecular pharming in cereal crops

2008

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There are many different agricultural expression systems that can be used for the largescale production of recombinant proteins, but fieldgrown cereal crops are among the most attractive because recombinant proteins can be targeted to accumulate in the seed, and specifically in the endosperm, which has evolved naturally as a protein storage tissue. Within the developing endosperm, proteins are supplied with molecular chaperones and disulfide isomerases to facilitate folding and assembly, while the mature tissue is desiccated to prevent proteolytic degradation. Proteins expressed in cereal seeds can therefore remain stable for years in ambient conditions. Recent basic research has revealed a surprising diversity of protein targeting mechanisms in the endosperm, which can help to control posttranslational modification and accumulation. Applied research and commercial development has seen several pharmaceutical proteins produced in cereals reach late stage preclinical development and the first clinical trials, with a number of companies now dedicated to developing cereal-based production platforms. In this review we discuss the basic science of molecular pharming in cereals, some of the lead product candidates, and challenges that remain to be addressed including the emerging regulatory framework for plant-made pharmaceuticals.

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Section: Downstream Processing and Purificationmentioning

confidence: 99%

Molecular pharming in cereal crops

2008

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“…The peptide extracts were allowed to pass through cation and anion exchange resin columns (Dowex 50 and Dowex 1, Sigma Chemical Co., USA) (Azzoni et al, 2005). 3N ammonia and 1N HCl was used for cation and anion exchange column respectively during peptide elution procedure.…”

Section: Ion Exchange Chromatographymentioning

confidence: 99%

ISOLATION, PURIFICATION AND PARTIAL CHARACTERIZATION OF LOW MOLECULAR WEIGHT PEPTIDES FROM NONPRIMED AND HALOPRIMED SEEDLINGS of Vigna mungo L. AND Cajanus cajan L. AND THEIR IMPACT ON PHYSIOLOGICAL ASPECTS UNDER NaCl EXPOSURE

Biswas¹,

Ghosh²,

Paul³

et al. 2019

JEBAS

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Plants confront an array of environmental stresses by tightly regulating their signalling pathways involving low molecular weight peptide synthesis. In the conducted study, initially growth parameters and ion contents were measured from salt stressed and haloprimed seedlings of blackgram (Vigna mungo L.) and pigeonpea (Cajanus cajan L.). Seedlings raised from nonprimed and haloprimed seeds were grown in hydroponic solution supplemented with desired concentration of NaCl for 3 weeks under controlled physiological conditions. Attempts were made to isolate low molecular weight peptide(s) from non-stressed, NaCl stressed and haloprimed seedlings of blackgram and pigeonpea, to quantify their variations and detect their molecular weight using HPLC and MALDI-TOF analysis respectively. Free radical scavenging activities of these peptides were studied under NaCl exposure. Subsequent bioassays were performed to determine the effect of these peptides on their respective physiological parameters. Peptide abundance was maximum in control seedlings of both the cultivars, which under NaCl stress became scanty. CD spectroscopic analysis confirmed reduced secondary conformations and more unordered peptides under NaCl stress. Haloprimed seedlings recovered such adversities to variable extents, resulting in improved germination and growth of test seedlings.

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“…The purification workhorse for many protein separation processes, including those published for purifying recombinant proteins from plants,19–21 is selective adsorption and elution of the target molecule using a packed bed of porous resin beads containing specific adsorptive ligands (i.e., ion exchange, hydrophobic, or affinity) 22, 23. The operation provides excellent purification factors, is reliably scaled between development and manufacturing sizes, and can be easily validated for commercial production 24.…”

Section: Introductionmentioning

confidence: 99%

Recovery of proteins from corn and soybean extracts by membrane adsorption

Menkhaus

Roseland²

2008

Biotechnology Progress

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in Wiley InterScience (www.interscience.wiley.com).Efficient separation strategies for the recovery of high-value proteins (native or recombinant) from plant agriculture are an important aspect of many different processes, from biopharmaceuticals to byproduct recovery during biofuel production. Here we report the use of membrane adsorption for the recovery of proteins from soybean and corn extracts, and compare the results with packed bed adsorption. Two alternative operating modes were investigated, a flowthrough strategy and a bind and elute method. Overall, membrane adsorption provided faster throughput, and had equal or slightly higher dynamic binding capacities compared with resin beads, without compromising yield and purity of the chosen target. Soybean was found to be an ideal plant host when capturing native protein on an anion exchange medium. This provided an opportunity to capture a large percentage ([80%) of native protein as the product, and/or allowed for elevated enrichment factors ([20) during purification of a recombinant target with pI [ 7.0, using a flowthrough approach. On the other hand, for corn, a single ion-exchange step was not able to capture more than 60% of the native protein. However, the bind and elute method with corn as the host for a recombinant product allowed for higher enrichment factors compared to soybean. In all cases, the concentration of a recombinant protein (as dictated by expression level) was found to play a significant role in the level of dynamic binding capacity, with higher concentration leading to elevated capacity. Likewise, a higher concentration of competing proteins was shown to decrease the overall capacity of a recombinant target.

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Purification of recombinant aprotinin produced in transgenic corn seed: separation from CTI utilizing ion-exchange chromatography

Cited by 10 publications

References 15 publications

Molecular pharming in cereal crops

Molecular pharming in cereal crops

ISOLATION, PURIFICATION AND PARTIAL CHARACTERIZATION OF LOW MOLECULAR WEIGHT PEPTIDES FROM NONPRIMED AND HALOPRIMED SEEDLINGS of Vigna mungo L. AND Cajanus cajan L. AND THEIR IMPACT ON PHYSIOLOGICAL ASPECTS UNDER NaCl EXPOSURE

Recovery of proteins from corn and soybean extracts by membrane adsorption

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