A novel carbodiimide coupling method for synthetic peptides

Deen, C.; Claassen, Eric; Gerritse, Koen; Zegers, N.D.; Boersma, W.J.A.

doi:10.1016/0022-1759(90)90428-x

Cited by 54 publications

(21 citation statements)

References 17 publications

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“…Dot blot immuno-assays, SDS-tricine-polyacrylamide gel electrophoresis (SDS-tricine-PAGE), blotting oiito membranes and Western analysis using enhanced chemiluminescence (ECL, Amersham, UK) and horse radish peroxidase-labelled secondary antibody were as described previously (Florack et al, 1994). Antiserum was raised in a rabbit against a synthetic cecropin B peptide (NH2-LysTrp-Lys-Val-Phe-Lys-Lys-Ile-Glu-Lys-Met Gly-ArgAsn-Ile-Arg-Asn-Gly-Ile-Val-Lys-Ala-Gly-Pro-Ala-IleAla-Val-Leu-Gly-Glu-Ala-Lys-Ala-Leu-COOH) (M r --3836), purchased from the American Peptide Co., Inc. (Santa Clara, USA), which was coupled to bovine serum albumin using a water-soluble Carbodiimide to increase antigenicity, prior to subcutaneous injection (Deen et al, 1990).…”

Section: Analysis Of Expression Of Cecropin B Genesmentioning

confidence: 99%

Expression of giant silkmoth cecropin B genes in tobacco

Florack

Allefs

Bollen

et al. 1995

Transgenic Research

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Cecropin B is a small antibacterial peptide from the giant silkmoth Hyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco via Agrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance to Pseudomonas solanacearum, the causal agent of bacterial wilt of many crops, and P. syringae pv. tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin B in vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.

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Section: Analysis Of Expression Of Cecropin B Genesmentioning

confidence: 99%

Expression of giant silkmoth cecropin B genes in tobacco

Florack

Allefs

Bollen

et al. 1995

Transgenic Research

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“…Western blot analysis [43] was performed using a polyclonal antiserum raised in a rabbit against HTHs, coupled to bovine serum albumin (BSA) to increase antigenicity [9]. Alkaline phosphatase-labelled goat anti-rabbit IgG was from Sigma.…”

Section: Hordothionin Expression and Processing Analysismentioning

confidence: 99%

Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting

et al. 1994

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Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 mumol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 mumol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP- and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.

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“…A comparison of the enzymatic synthesis of the conjugates and of the chemical methods (see for example 15,16) shows that the new technique is less time consuming and needs less labor. The synthesis of the hapten conjugates by MTGase catalysis takes only 2 h, whereas chemical procedures require additional activation and purification steps.…”

Section: Resultsmentioning

confidence: 98%