Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris

Baumgartner, Philippe; Raemaekers, Romaan J. M.; Durieux, Alain; Gatehouse, A. M. R.; Davies, Howard V.; Taylor, Mark A.

doi:10.1016/s1046-5928(02)00555-7

Cited by 39 publications

(17 citation statements)

References 17 publications

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“…The garlic lectins could be produced as functional recombinant proteins in P. pastoris in adequate quantities for insect feeding trial experiments, although yields (10-20 mg l À1 ) were lower than those reported for other plant lectins expressed in Pichia, such as the kidney bean (Phaseolus vulgaris) agglutinin E-form (PHA-E, 100 mg l À1 ); and snowdrop lectin (GNA, 80 mg l À1 ) (Baumgartner et al, 2002(Baumgartner et al, , 2003. Besides the expected removal of the N-terminal prepro-sequence of the yeast a-factor from the recombinant proteins, C-terminal post-translational processing of both recombinant lectins took place either in P. pastoris, or after secretion to the medium.…”

Section: Discussionmentioning

confidence: 92%

The insecticidal activity of recombinant garlic lectins towards aphids

Fitches¹,

Wiles

Douglas

et al. 2008

Insect Biochemistry and Molecular Biology

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Section: Discussionmentioning

confidence: 92%

The insecticidal activity of recombinant garlic lectins towards aphids

Fitches¹,

Wiles

Douglas

et al. 2008

Insect Biochemistry and Molecular Biology

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“…The filter-based colony immunodetection method, termed ''yeastern blot'' [28], was used as a preliminary screen to semi-quantitatively detect the clones expressing the highest levels of recombinant avidin.…”

Section: Screening Transformants For High-level Expressionmentioning

confidence: 99%

Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris

Zocchi

Jobé

Neuhaus

et al. 2003

Protein Expression and Purification

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A recombinant glycosylated avidin (recGAvi) with an acidic isoelectric point was expressed and secreted by the methylotrophic yeast Pichia pastoris. The coding sequence for recGAvi was de novo synthesized based on the codon usage of P. pastoris. RecGAvi is secreted at approximately 330 mg/L of culture supernatant. RecGAvi monomer displays a molecular weight of 16.5 kDa, as assessed by ESI mass spectrometry. N-terminal amino acid sequencing indicates the presence of three additional amino acids (E-A-E), which contribute to further lowering the isoelectric point to 5.4. The data presented here demonstrate that the P. pastoris system is suitable for the production of recGAvi and that the recombinant avidin displays biotin-binding properties similar to those of the hen-egg white protein.

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“…The galactose‐binding lectin frutalin from breadfruit seeds was successfully expressed at 18–20 mg·L −1 [16]. Much higher yields of recombinant protein can be obtained when Pichia cultures are grown in a bioreactor under controlled conditions, as reported for the recombinant lectins from Aleuria aurantia (67 mg·L −1 ) [22], snowdrop (80 mg·L −1 ) [23] and the bean lectin PHA‐E (100 mg·L −1 ) [24].…”

Section: Discussionmentioning

confidence: 99%

Expression analysis of the nucleocytoplasmic lectin ‘Orysata’ from rice in Pichia pastoris

et al. 2011

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The Oryza sativa lectin, abbreviated Orysata, is a mannose‐specific, jacalin‐related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin‐related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomycesα‐factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N‐glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high‐mannose structures, Man9–11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high‐mannose as well as with more complex N‐glycan structures. Orysata has potent anti‐human immunodeficiency virus and anti‐respiratory syncytial virus activity in cell culture compared with other jacalin‐related lectins.

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Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris

Cited by 39 publications

References 17 publications

The insecticidal activity of recombinant garlic lectins towards aphids

The insecticidal activity of recombinant garlic lectins towards aphids

Expression and purification of a recombinant avidin with a lowered isoelectric point in Pichia pastoris

Expression analysis of the nucleocytoplasmic lectin ‘Orysata’ from rice in Pichia pastoris

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