Protection of Mice Against a Lethal Influenza Challenge by Immunization with Yeast‐Derived Recombinant Influenza Neuraminidase

Martinet, Wim; Saelens, Xavier; Deroo, Tom; Neirynck, Sabine; Contreras, Roland; Jou, Willy Min; Fiers, Walter

doi:10.1111/j.1432-1033.1997.00332.x

Cited by 75 publications

(43 citation statements)

References 39 publications

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“…±, Not detected (the inhibition observed was not sufficient to calculate a concentration of the inhibitor); ND, not determined. the glutamic acid-alanine sequence from the N-terminus has been reported for other proteins [34].…”

Section: A Yeast Expression System For Pi-2mentioning

confidence: 99%

Characterization of potato proteinase inhibitor II reactive site mutants

Beekwilder

Schipper

Bakker

et al. 2000

European Journal of Biochemistry

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Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (K i 0.4 nm) and chymotrypsin (K i 0.9 nm), and is quite robust towards mutations at positions P2 or P1H . In contrast, the first reactive site inhibits only chymotrypsin (K i 2 nm), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.Keywords: mutational analysis; Pichia pastoris; serine proteinase inhibitor; Solanum tuberosum.Protease inhibitors are abundant proteins in the storage organs and seeds of plants [1]. In addition, their synthesis is induced to high levels in response to stress, infection and wounding [2]. Potato expresses many different proteinase inhibitors belonging to a wide range of inhibitor families. Members of the potato proteinase inhibitor II (PI-2) family have been shown to inhibit serine proteases, such as trypsin, chymotrypsin, subtilisin, oryzin and elastase [3,4]. Until now, they have been found only among the Solanaceae. Proteins and messenger RNAs have been identified in potato tubers [5,6], wounded tomato and tobacco leaves [4,7], eggplant fruits [8], green tomatoes [9] and ornamental tobacco flower stigma [10]. There is medical interest in the properties of PI-2. It has been shown to have anticarcinogenic properties, protecting mouse embryo fibroblasts from radiation-induced transformation [11]. Although the mechanism underlying this effect is poorly understood, it is correlated to the capacity of PI-2 to inhibit chymotrypsin-like enzymes [12].Plant protease inhibitors such as PI-2 have been proposed to function as part of the plant defense system [13]. The plant defense role is deduced from observations that proteinase inhibitors in leaves are synthesized in response to wounding [2,14,15] or viral infection [2,16]. In addition, direct evidence for a protective role has been obtained; it was shown that transgenic tobacco plants over-expressing potato PI-2 gained resistance against the tobacco hornworm Manduca sexta [17]. Also, when the PI-2 gene was introduced into rice under its own wound-inducible promoter, plants were protected from the major lepidopteran rice pest, Sesamia inferens [18]. PI-2 probably acts by inhibiting digestive proteases from the insects, thereby restricting the availability of amino acids [13].The anti-nutrient activity of PI-2 has been shown to be overcome by pest insects like Spodoptera exigua [19]. When these larvae are reared on tobacco leaves expressing potato PI-2, the spe...

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Section: A Yeast Expression System For Pi-2mentioning

confidence: 99%

Characterization of potato proteinase inhibitor II reactive site mutants

Beekwilder

Schipper

Bakker

et al. 2000

European Journal of Biochemistry

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“…The major advantages of this expression system include (i) a strong, tightly regulated alcohol oxidase (AOX) promoter, 5ЈAOX1, is available; (ii) large-scale protein production can be achieved in a large-volume fermentor culture; (iii) a secretary pathway allows the product to be secreted into the medium, separating the foreign protein from most of the host proteins; (iv) the expression system can be easily set up; and (v) the cost is as low as that of the E. coli expression system (2,4,6,23). Two membrane proteins of influenza virions, neuraminidase (NA) and hemagglutinin (HA), were successfully expressed in P. pastoris and produced as secreted forms in the culture medium (24,37). These influenza virus proteins served as recombinant vaccines that elicit partial or fully protective antibodies in mice.…”

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Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

Hwang

Yamada

Honda

et al. 2000

J Virol

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Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni 2؉ -agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris. Influenza A virus contains eight different RNA segments of negative polarity in its genome, each encoding one or two unique viral proteins. The viral RNA polymerase is associated with each RNA segment as a viral component and in infected cells, responsible for both transcription and replication of the viral genome (for reviews, see references 7, 13, and 21). The pathway for the synthesis of viral mRNA involves multiple-step reactions, consisting of endonucleolytic cleavage of host cell mRNA, capped oligonucleotide-primed transcription of viral RNA (vRNA), and the addition of a poly(A) tail to the nascent mRNA. On the other hand, replication takes place in two steps, vRNA-directed synthesis of full-length complementary RNA (cRNA) without any modification at both the 5Ј and 3Ј termini and cRNA-directed reproduction of vRNA.Starting from the viral ribonucleoprotein (RNP), we isolated an RNA-3P (PB1, PB2, and PA) protein complex without NP by equilibrium centrifugation in cesium sulfate or cesium chloride (15, 16). The RNA-3P complex is enzymatically active in the in vitro synthesis of short attenuated RNA chains (but NP is needed for RNA chain elongation [12]), indicating that the three P proteins participate in the catalytic function of RNA polymerization. The vRNA-free 3P complex, consisting of one molecule each of the three P proteins, was isolated from the RNA-3P complex by centrifugation in cesium chloride or cesium trifluoroacetate (11). The 3P complexes exhibited RNA synthesis only when a model vRNA template with 5Ј-and 3Ј-terminal cons...

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“…NA immunogenicity was first observed in human subjects immunized with an NA-specific inactivated vaccine (16). The use of recombinant NA (rNA) proteins expressed in yeast (17) or insect cells (18) elicits protection against lethal virus challenges in immunized mice. Ferrets immunized with rNA proteins exhibit a distinctive type of protection in addition to that provided by HA immunization alone (19).…”

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confidence: 99%