Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties

Romanos, Michael A.; Clare, Jeffrey J.; Beesley, Katrina M.; Rayment, Fred B.; Ballantine, Stuart P.; Makoff, Andrew; Dougan, Gordon; Fairweather, Neil F.; Charles, Ian G.

doi:10.1016/0264-410x(91)90011-t

Cited by 88 publications

(27 citation statements)

References 20 publications

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“…3). The expression yield (1.57 g/ml) of 3P was not as high as those of several heterologous proteins, such as human tumor necrosis factor (42), the antigen pertactin (p69) of Bordetella pertussis (35), and glycosylated invertase (47), which have all been expressed in P. pastoris up to several grams per liter of culture. However, the expression levels of P proteins are as high as those of two membrane proteins of influenza virus, neuraminidase (NA) and hemagglutinin (HA), which ranged from 2.5 to 3 g/ml (24) and 0.375 to 0.675 g/ml (37), respectively.…”

Section: Discussionmentioning

confidence: 97%

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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Section: Discussionmentioning

confidence: 97%

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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“…Ninety-one His ϩ transformants were tested for gene integration into the Pichia genome using a rapid DNA dot-blot technique (25). There were seven transformants that exhibited strong hybridization signals.…”

Section: Resultsmentioning

confidence: 99%

“…His ϩ transformants in KM71 were purified on regeneration dextrose plates (Invitrogen) without histidine. Gene integration was detected using a rapid DNA dot blot technique (25). To express the enzyme, a single colony of multiple integration transformants was inoculated into 25 ml of pH 6-buffered complex glycerol media (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Riboflavin 5′-Hydroxymethyl Oxidation

Chen

McCormick

1997

Journal of Biological Chemistry

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Vitamin B 2 -aldehyde-forming enzyme catalyzes oxidation of the 5-hydroxymethyl of riboflavin to the formyl group. We have purified the enzyme from the culture media of Schizophyllum commune (ATCC 38719) by modifying the procedure of Tachibana and Oka (Tachibana, S., and Oka, M. (1981) J. Biol. Chem. 256, 6682-6685) for cell-free extract. By SDS-polyacrylamine gel electrophoresis, the enzyme appears to be 78 kDa. The enzyme has a blocked amino terminus, so fragments were obtained by cleaving the purified enzyme with lysyl endopeptidase. Selected peptides were sequenced from their amino termini. We have isolated a full-length cDNA clone using a DNA hybridization probe amplified by polymerase chain reaction with two degenerate oligonucleotide primers, the design of which was based on one of the partial amino acid sequences. From the cDNA clone, it is evident that the enzyme has a Ser/Thr-rich fragment near the COOH-terminal Asp. The enzyme was determined to be a glycoprotein; however, O-deglucosylation only slightly affects activity. Computer searches showed that the B 2 -aldehyde-forming enzyme has little homology with other proteins, but domain motifs may reflect N-myristoylation of a dehydrogenase with a signature similar to 4Fe-4S ferredoxins. The enzyme cDNA was subcloned into a Pichia expression vector pPIC9K to produce a recombinant protein which exhibited B 2 -aldehyde-forming enzyme activity.The metabolism of riboflavin is better understood in terms of enzymes responsible for the biosynthetic conversion of this vitamin to its functional coenzymes than as concerns enzymes catalyzing catabolic degradation of the vitamin (1). Complete purification of flavokinase (2), which catalyzes formation of FMN, and FAD synthetase (3), have led to significant understanding of flavocoenzyme formation and its kinetic control (4); however, not one of the enzymes responsible for degradation of riboflavin had been sequenced or obtained in sufficient quantity to allow suitable molecular characterization.The considerable extent to which riboflavin is apparently catabolized in the human was reflected by early balance experiments in adult men (5). Although diverse flavin catabolites have been described in nature (6), little has been done to elucidate the enzyme systems responsible for their formation. Certain bacteria have been found able to degrade the flavin isoalloxazine ring (7, 8) as well as D-ribityl chain (9, 10) and its analogues (11). Oxidations of flavin 7-and 8-methyl functions lead to the corresponding hydroxymethyl compounds found in urine (12-14) and milk (15, 16) and to 7␣-hydroxymethyl-riboflavin as the main catabolite in human plasma (17). The enzyme system responsible for forming so-called "schizoflavins", which are the aldehyde and carboxylic acid derived from oxidation of the terminal 5Ј-hydroxymethyl of the riboflavin side chain (18), was purified from the fungus Schizophyllum commune by Tachibana and Oka (19,20). Our laboratory first ascertained the rather narrow flavin specificity of the S. commune ribofla...

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“…Another way of generating multicopy strains is simply to screen large numbers of colonies for spontaneously occurring transformants with higher gene dosage or higher product expression (Clare et al, 1991a;Romanos et al, 1991;Wung and Gascoigne, 1996). However, at this time only two dominant markers, the bacterial Sh ble (Zeocin r ) or BSD (blasticidin r ) genes, can be used to directly select transformants and screen for high-copy strains on increasingly higher concentrations of Zeocin or blasticidin (Miles et al, 1998;Romanos et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

“…However, at this time only two dominant markers, the bacterial Sh ble (Zeocin r ) or BSD (blasticidin r ) genes, can be used to directly select transformants and screen for high-copy strains on increasingly higher concentrations of Zeocin or blasticidin (Miles et al, 1998;Romanos et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

Direct selection of Pichia pastoris expression strains using new G418 resistance vectors

Lin‐Cereghino

Hashimoto

Moy

et al. 2008

Yeast

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The methylotrophic yeast, Pichia pastoris, is widely used as a host organism for the expression of heterologous proteins. Currently, the Zeocin and blasticidin resistance genes are the only dominant selectable markers that can be used for primary selection of transformants. In this report we describe new expression vectors that can be used to select directly for P. pastoris transformants using G418 resistance conferred by a modified Tn903kan r gene. Compared to other dominant markers, this system is more economical and offers a higher transformation efficiency, due to the small sizes of the cloning vectors, pKAN B and pKANα B (GenBank Accession Nos EU285585 and EU285586, respectively). Additionally, multicopy transformants can be generated using these new vectors.

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Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties

Cited by 88 publications

References 20 publications

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

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

Riboflavin 5′-Hydroxymethyl Oxidation

Direct selection of Pichia pastoris expression strains using new G418 resistance vectors

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