Effect of Copy Number on the Expression Levels of Hepatitis B Surface Antigen in the Methylotrophic Yeast Pichia pastoris

Vassileva, Ana; Chugh, Dipti A.; Swaminathan, Sathyamangalam; Khanna, Navin

doi:10.1006/prep.2000.1335

Cited by 136 publications

(105 citation statements)

References 21 publications

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“…Sistem ini dapat menghasilkan ekspresi yang tinggi dengan regulasi ketat menggunakan induksi metanol pada promoter alkohol (AOX1) [Cheregino et al 2000]. Oleh karena itu, merupakan suatu keuntungan untuk mengembangkan sistem P.pastoris yang ekonomis untuk produksi vaksin Hepatitis B dalam skala besar [Bo et al 2005, Vassileva et al 2001.…”

Section: Pendahuluanunclassified

Budaya Ekologi Suku Talang Mamak Dalam Pengelolaan Hutan

Yunus¹

2017

Preprint

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AbstrakDegradasi lingkungan yang terjadi selama ini bermuara kepada manusia, baik sebagai penyebab maupun sebagai penerima dampak. Kajian mengenai praktik-praktik berkelanju- tan yang dilaksanakan dengan mengintegrasikan antara budaya dan ekologi mutlak diper- lukan, salah satunya Suku Talang Mamak. Penelitian ini dilaksanakan di Desa Talang Ged- abu, Kecamatan Rakit Kulim, Kabupaten Indragiri Hulu, Provinsi Riau pada bulan Oktober 2016 sampai Januari 2017. Penelitian ini dilakukan dengan metode survei, wawancara, Fo- cus Group Discussion (FGD) dan studi pustaka. Data yang diperoleh kemudian dianalisis secara deskriptif untuk mengungkap, menelaah dan memahami gejala-gejala dalam penelitian. Komponen pengelolaan meliputi: (a) perencanaan; (b) pemanfaatan; (c) pengen- dalian; (d) pemeliharaan; (e) pengawasan; (f) penegakan hukum. Budaya ekologi Suku Ta- lang Mamak dalam pengelolaan hutan mengandung berbagai nilai-nilai yang meliputi pengetahuan lokal dalam aspek perencanaan. Pemanfaatan sumber daya hutan dilakukan dengan mempertimbangkan keberlanjutan, fungsi dan produktivitas hutan. Pengendalian meliputi upaya pencegahan, penanggulangan dan pemulihan melalui pantang larang dengan kendali pimpinan adat. Pemeliharaan meliputi upaya konservasi, pencadangan dan peles- tarian hutan melalui sistem kerja gotong royong dan kepercayaan akan mitos dan adanya hukum adat yang mengatur tentang keberadaan tanah keramat.Kata kunci: Adat, Budaya ekologi, Pengelolaan hutan, Suku Talang Mamak AbstractEnvironmental degradation that occurred during this time leads to humans, both as the cause and as the recipient of the impact. A study of sustainable practices implemented by integrating culture and ecology is absolutely necessary, one of which is Suku Talang Mamak. This research was conducted in Talang Gedabu Village, Rakit Kulim Sub-district, Indragiri Hulu Regency, Riau Province from October 2016 until January 2017. This research was conducted by survey method, interview, Focus Group Discussion (FGD) and literature study. Data obtained then analyzed descriptively to reveal, review and understand the symptoms in the study. Management components include: (a) planning; (b) utilization; (c) control; (d) maintenance; (e) supervision; (f) law enforcement. The ecological culture of Suku Talang Mamak in forest management contains various values that include local knowledge in the planning aspect. Utilization of forest resources is carried out by considering the sustaina- bility, function, and productivity of forests. Control includes prevention, mitigation, and re- covery through prohibition with the control of adat leaders. Maintenance includes conser- vation, reserve and forest conservation through a system of mutual cooperation and belief Bunga Rampai Forum Peneliti Muda Indonesia 2017in the myth and the existence of customary law governing the existence of sacred land (tanah keramat).Keywords: Customs, Cultural ecology, Forest management, Talang Mamak

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Section: Pendahuluanunclassified

Budaya Ekologi Suku Talang Mamak Dalam Pengelolaan Hutan

Yunus¹

2017

Preprint

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“…The strain is a histidine requiring auxotroph that has an intact AOX1 gene and is capable of utilizing methanol efficiently (Vassileva et al, 2001). Single and multicopy HBsAg expression cassettes were integrated into the P. pastoris genome in the AOX locus using a gene replacement strategy.…”

Section: Constructionmentioning

confidence: 99%

“…Single and multicopy HBsAg expression cassettes were integrated into the P. pastoris genome in the AOX locus using a gene replacement strategy. Prior to transformation, Indonesian HBsAg expression plasmids were cleaved with BglII, resulted one of the fragment contained of Indonesian HBsAg expression cassette and His4 selection marker which are flanked by sequences homologues to the 59 and 39 ends of the AOX1 locus (Vassileva et al, 2001). The fragments were subsequently purified and used for further transformation.…”

Section: Constructionmentioning

confidence: 99%

“…This system could generate highly expressed heterologous protein with a strong and tightly regulated methanol-inducible alcohol oxidase (AOX1) promoter (Cereghino and Cregg, 2000). Therefore, it would be advantageous to develop P. pastoris as an inexpensive expression system for the production of hepatitis B vaccine in large quantities (Bo et al, 2005;Vassileva et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

“…The increase in copy number of s-HBsAg expression cassette in P. pastoris GS115 results in a proportional elevation in the steady state levels of the s-HBsAg-specific mRNA, which in turn increase the total levels of the s-HBsAg protein (Vassileva et al, 2001). Based on those results, we regenerated new recombinant multi Indonesian s-HBsAg expression cassettes which was designed and synthesized based on the most HBV genotype and serotype found in Indonesian areas, B3 genotype and adw serotype (Narita et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Construction, Expression and Characterization of Multi Cassettes Encoding Indonesian Small Hepatitis B Surface Antigen (s-HBsAg) in Methylotropic Yeast Pichia pastoris

Rachman¹,

Utami²,

Kusumaward³

et al. 2015

Biotechnology

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A B S T R A C T Hepatitis B is the most common liver infection worldwide. The recombinant VirusLike Particles (VLP) of small Hepatitis B surface antigen (s-HBsAg) vaccine provides excellent protection against Hepatitis B Virus (HBV). In this study, we have generated a construct of Pichia pastoris recombinant which comprises multi expression cassettes encoding s-HBsAg which belongs to HBV B3 genotype predominantly found in Indonesia. An expression cassette containing a synthetic codon optimized open reading frame of HBV B3 genotype/adw serotype s-HBsAg was cloned into pAO815 to generate pAOHBs1 in Escherichia coli. The expression cassette from pAOHBs1 was subsequently isolated and ligated with recombinant previously obtained, generated recombinant plasmid containing 2, 3 and 4 direct repeats of HBsAg expression cassette. The resulted recombinant containing 4 expression cassettes was integrated to P. pastoris genome. Positive integrants and stability during cultivation were identified by PCR and qPCR. The expression of s-HBsAg was induced by methanol and analyzed by SDS PAGE and western blotting. Nucleotide sequencing analysis showed that the inserted fragment encodes amino acids with 100% similarity compared to that designed for HBV B3 genotype. The PCR and qPCR analysis showed that stable P. pastoris integrated with 4 s-HBsAg expression cassette was successfully obtained with methanol utilizing slow phenotype. A protein band with apparent molecular weight which similar to s-HBsAg size was detected based on a SDS PAGE and western blot analysis. A stable integrant of P. pastoris containing four expression cassettes of HBV B3 genotype s-HBsAg capable of producing a vaccine candidate against Hepatitis B was generated.

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Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae

Hemayatkar¹,

Mahboudi²,

Majidzadeh‐A³

et al. 2010

Biotechnology Journal

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Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.

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Effect of Copy Number on the Expression Levels of Hepatitis B Surface Antigen in the Methylotrophic Yeast Pichia pastoris

Cited by 136 publications

References 21 publications

Budaya Ekologi Suku Talang Mamak Dalam Pengelolaan Hutan

Budaya Ekologi Suku Talang Mamak Dalam Pengelolaan Hutan

Construction, Expression and Characterization of Multi Cassettes Encoding Indonesian Small Hepatitis B Surface Antigen (s-HBsAg) in Methylotropic Yeast Pichia pastoris

Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae

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