Characterization of the Erwinia carotovora pelB gene and its product pectate lyase

Lei, Shau-Ping; Lin, Hun-Chi; Wang, Shanshan; Callaway, J. C.; Wilcox, Gary

doi:10.1128/jb.169.9.4379-4383.1987

Cited by 248 publications

(119 citation statements)

References 13 publications

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“…Based on refs. [30,31], the HRP gene was directionally cloned into the pQE60 vector as a fusion with the N-terminal pectate lyase (PelB) leader sequence [32] and a Cterminal hexa-histidine purification tag, to generate the plasmid pBR_I. Initially, the PelB leader was cloned via a Nco I -BamH I double restriction.…”

Section: Methodsmentioning

confidence: 99%

Effects of mutations in the helix G region of horseradish peroxidase

Ryan¹,

Ó’Fágáin²

2008

Biochimie

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Summary.Horseradish Peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its usefulness. Based on prior art, we substituted solvent-exposed lysine and glutamic acid residues near the proximal helix G (Lys 232, 241; Glu 238, 239) and between helices F and F' (Lys 174). Three single mutants (K232N, K232F, K241N) demonstrated increased stabilities against heat (up to two-fold) and solvents (up to four-fold). Stability gains are likely due to improved hydrogen bonding and space-fill characteristics introduced by the relevant substitution. Two double mutants showed stability gains but most double mutations were non-additive and non-synergistic.Substitutions of Lys 174 or Glu 238 were destabilising. Unexpectedly, notable alterations in steady-state V m /E values occurred with reducing substrate ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)), despite the distance of the mutated positions from the active site.

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Section: Methodsmentioning

confidence: 99%

Effects of mutations in the helix G region of horseradish peroxidase

Ryan¹,

Ó’Fágáin²

2008

Biochimie

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“…The constructs are designed to include the widely used Erwinia carotovora pelBleader peptide 21 for targeted translocation of the scFv peptide chain to the periplasmic space at the translation start (Fig. 1).…”

Section: Assessment Of a New Scfv Production Hostmentioning

confidence: 99%

Biomedicals from a soil bug

Dammeyer

2012

Bioengineered

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“…The genetic construct encodes the pelB leader sequence (Lei et al, 1987) that directs the expressed protein to the bacterial periplasm, where the oxidising environment should permit the formation of intramolecular disulphide bonds. Whilst the majority of material produced by the bacteria was degraded by proteases to give a product similar to the native SCA, a significant quantity of functional material was obtained from the culture supernatant and partially purified by affinity chromatography.…”

Section: Discussionmentioning

confidence: 99%

A recombinant single-chain antibody interleukin-2 fusion protein

Savage

Spooner

et al. 1993

Cell Biophysics

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SummaryRecombinant interleukin-2 (rIL-2) therapy has been shown to be of value in the treatment of some cases of melanoma and renal cell carinoma. However its use can be limited by severe systemic toxicity. Targeting rIL-2 to the tumour should improve the anti-tumour immune response and decrease the systemic toxicity. With this aim we have employed recombinant DNA techniques to construct a single chain antibody interleukin-2 fusion protein (SCA-IL-2).The protein used in this model system comprises the variable domains of the anti-lysozyme antibody D1.3 fused to human IL-2. It has been expressed by secretion from Escherichia coli and the purified product possesses antigen binding specificity and retains the immunostimulatory activities of rIL-2.This approach can be taken to generate SCA-IL-2 proteins that bind to appropriate cellular antigens. In vivo administration of a tumour binding SCA-IL-2 should result in a localised high concentration of IL-2 in tumour tissues, maximising the anti-tumour immune response, whilst keeping systemic side effects to a minimum.

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Characterization of the Erwinia carotovora pelB gene and its product pectate lyase

Cited by 248 publications

References 13 publications

Effects of mutations in the helix G region of horseradish peroxidase

Effects of mutations in the helix G region of horseradish peroxidase

Biomedicals from a soil bug

A recombinant single-chain antibody interleukin-2 fusion protein

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