Expression and characterisation of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat

Longstaff, Marian; Newell, C. A.; Boonstra, Birgitte; Strachan, Gillian; Learmonth, D; Harris, William J.; Porter, A.J.; Hamilton, W. D.

doi:10.1016/s0304-4165(98)00024-5

Cited by 44 publications

(13 citation statements)

References 34 publications

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“…Longstaff et al [59] described the functional expression of xenobiotics-specific antibody fragments in transgenic plants of Nicotiana tabacum cv. Samsun NN and cell suspension cultures.…”

Section: Choice Of Expression Hostmentioning

confidence: 99%

Recombinant antibodies for environmental analysis

Kramer

Hock

2003

Analytical and Bioanalytical Chemistry

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Initial steps of antibody engineering in the late eighties revolutionized the technology of antibody production, particularly in the area of immunotherapy and diagnostics. Hallmarks that seemed to be out of reach for a long time are now the state of the art, e.g. tailoring of antibodies to match particular needs or by-passing immunization by use of antibody libraries. Despite the apparent benefits of recombinant antibody technologies, this field has been opened up hesitantly for other applications. This review addresses the development of recombinant antibody synthesis in environmental analysis. Examples are given of the molecular evolution of pesticide antibodies and their application for the analysis of real samples.

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“…Longstaff et al [59] described the functional expression of xenobiotics-specific antibody fragments in transgenic plants of Nicotiana tabacum cv. Samsun NN and cell suspension cultures.…”

Section: Choice Of Expression Hostmentioning

confidence: 99%

Recombinant antibodies for environmental analysis

Kramer

Hock

2003

Analytical and Bioanalytical Chemistry

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“…Thus, an accurate and rapid method is needed to detect this herbicide. Immunoassays that employ polyclonal antibodies (pAb), mAb (Eremin et al, 1994), or rAb fragments (Ward et al, 1993;Kramer and Hock, 1996;Longstaff et al, 1998) have been developed to facilitate the detection of atrazine, but the conventional production of large amounts of pesticide-specific antibodies is expensive and requires appropriate conjugates consisting of a small analyte and a large molecule for the immunization of the animals. In contrast, large-scale production of rAb fragments in microorganisms is inexpensive and their properties can be manipulated as desired by genetic engineering.…”

Section: Introductionmentioning

confidence: 99%

High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris

Lange

Schmitt

Schmid

2001

Journal of Immunological Methods

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In this report, we describe the high-yield secretory expression (~ 40 mg l -1 ) of pure, atrazinespecific Fab fragments (K411B) from P. pastoris that was achieved by co-integration of the genes encoding the heavy and light chains ( Crude culture supernatant was used to study the binding properties of the Fab fragment toward different s-triazines by means of competitive ELISA: the IC 50 value for the detection of atrazine was determined from the standard curve as 3 µg l -1 , which is one magnitude higher than the value obtained with the parental mAb K4E7 but equals that obtained when the same Fab fragment was expressed in E. coli cells. In addition, the cross-reactivity pattern of the Fab from Pichia is comparable to that of E. coli and the parental mAb K4E7.

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“…The codons ending with CG/TA were independent T 0 transgenic tomato plants (PAK 1,3,5,11,16,23,24,29,30,31 and 32) avoided, six putative polyadenylation signals, five mRNA instability sequences and potential splicing sites were eliminated while designing the modified AAT gene. The 24-amino acid signal peptide of native AAT was replaced either with tobacco PR1a signal peptide (Longstaff et al 1998), sweet potato sporamineA (Matsuoka and Nakamura 1991) or with dicot-preferred native signal peptide sequence of the gene and an ER-retention signal (KDEL) was introduced at the C-terminus for optimum targeting, glycosylation and accumulation of rAAT in leaves of transgenic tomato plant. A dicot preferred translation initiation context (TAAACAATGG) sequence (Joshi et al 1997) and a 38 bp AMV 5 0 UTR was incorporated at the 5 0 end of the modified gene for efficient and optimum expression of the recombinant protein.…”

Section: Discussionmentioning

confidence: 99%

Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

et al. 2008

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Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.

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Expression and characterisation of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat

Cited by 44 publications

References 34 publications

Recombinant antibodies for environmental analysis

Recombinant antibodies for environmental analysis

High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris

Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

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