Annales Bogorienses

Number 1, 2014

13-23
Construction and Expression of Immunotoxin Anti EGFRvIII scFv-HPR Conjugate in Pichia pastoris as A Targeted Drug Candidate for Cancer Therapy

Authors: Yuliawati Yuliawati, Retno Damayanti Soejoedono, Asrul Muhamad Fuad

Abstract

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Epidermal growth factor receptor variant III (EGFRvIII) is a mutant of EGFR lacking 267 amino acids (exon-2 through 7) within its extracellular domain, resulting in the formation of a new epitope as a tumorspecific target. EGFRvIII is commonly found in GBM, breast, ovarian, prostate, and lung carcinomas. Antibodies or part of antibodies (e.g. single chain variable fragment or scFv) with specific activity against this receptor have been developed. These antibodies are internalized into the cell after receptor binding. Ribonucleases can be cytotoxic due to their inherent ability to degrade RNA, therefore causing cell death via inhibition of protein synthesis. The aim of this research is to construct, clone and express an immunotoxin-based conjugate combining an anti-EGFRvIII scFv with a HPRmut (human pancreatic ribonuclease mutant variant) in Pichia pastoris. The anti-EGFRvIII scFv gene was cloned into yeast expression vector pPICZαA and fused with EGFP gene as a marker. The HPRmut gene was subsequently cloned at the C-terminal of the scFv::EGFP fusion, resulting in the scFv::EGFP::HPR fusion construct. The fusion construct was successfully obtained and nucleotide sequence of plasmid was verified. This construct was used to transform Pichia pastoris SMD 1168H. The gene fusion was successfully transformed and expressed in P. pastoris with a transformation efficiency of 102 cfu/μg DNA. The transformed yeasts were screened on agar media containing up to 1000 μg/ml zeocin. Yeast transformants showed green fluorescent due to the expression of EGFP gene. The recombinant proteins have been expressed and secreted from P. pastoris as showed by immunoblotting and SDS-PAGE analyses, then purified by affinity chromatography method. The selected yeast transformant produced at least 15.85 mg of purified protein per liter of yeast culture.

25-34
Expression of An Immunogenic Intimin Fragment of EHEC O157:H7 in Escherichia coli Periplasm under The Control of A Rhamnose-Based Regulated Promoter

Authors: Hariyatun Hariyatun, Antonius Suwanto, Wien Kusharyoto

Abstract

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Intimin is the main adhesin of Enterohemorrhagic E. coli (EHEC) O157:H7 bacteria which are the most common leading infectious cause of bloody diarrhea and acute kidney failure in children who develop hemolytic uremic syndrome (HUS). Intimin is required for persistent bacterial colonization to eukaryotic host cell and its receptor-binding activity is localized at the C-terminus 282 amino acids (Intimin282). Thus, Intimin282 is an attractive antigen candidate that could be useful in vaccine and diagnostic systems against EHEC infections. Previous studies had reported expression of Intimin in E. coli cytoplasm using commonly used prokaryotic expression systems. However, it usually encountered several problems, i.e. low expression level, leaky expression, inclusion body formation, and truncated protein. The pRHA vector, which is tightly regulated by Lrhamnose and D-glucose, represents a viable alternative E. coli expression system to overcome such problems. Moreover, E. coli periplasm has an advantage of maintaining protein functionality by providing an oxidative environment that is more efficient than cytoplasm. However, to date there is no study about Intimin expression using pRHA expression system and/or in E. coli periplasm. Accordingly, we constructed a recombinant pRHA vector harbouring the respective gene to investigate the expression of an immunogenic Intimin fragment of EHEC O157:H7 in E. coli periplasm. The gene encoding His6-tagged Intimin282 (Int282) together with pelB signal sequence was cloned into the pRHA vector, subsequently expressed in E. coli JM109 and purified. Expression and purification of Int282 were verified by SDS-PAGE and Western blot. The result showed that Int282 was successfully expressed in E. coli periplasm with a protein size of approximately 32 kDa, which corresponded with the predicted size of the protein based on its amino acid sequence.

35-43
Somatic Embryo Germination of Jatropha curcas L in Presence of Sucrose and Poly Ethylene Glycol (PEG)

Authors: Rudiyanto Rudiyanto, Darda Efendi, Tri Muji Ermayanti

Abstract

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Jatropha curcas L. is a potential source of a non-edible biofuel. Conventional propagation of J. curcas technique has some limitations. Somatic embryo can produce a large number of embryos and obtain a large number of plants all year round. Treatment of sucrose in combination with polyethylene glycol (PEG) was proven to enhance germination of somatic embryos in many plant species. The aim of the study was to investigate the effect of sucrose in combination of PEG on somatic embryo germination in J. curcas. Globular somatic embryos at 0.025-0.030 g fresh weight having 0.4-0.5 cm in diameter were grown on MS medium solidified with 3 g/l of Gelzan supplemented with sucrose at 20, 30, 40, and 50 g/l in combination with PEG at 0, 2.5, 5, 10, and 15%. Results showed that the best medium for germination of J. curcas somatic embryo cultures was MS medium supplemented with 20 and 30 g/l of sucrose in combination with 5% of PEG. The numbers of germinated embryos per clump had significant enhancement on those medium compared with the control (PEG free treatment) (2.65 to 5.65) and (2.55 to 5.50). In addition, those treatments resulted in the highest percentage of clumps forming germinated embryos (100%), with an average of normal germinated embryos at 94.163 and 96.065%. The addition of 40 and 50 g/l of sucrose in combination with 15% of PEG caused all embryos to fail at germinating.

45-53
In vitro Seed Germination and Shoot Multiplication of Seven Endemic Subalpine and Alpine Plant Species Grown on Mount Jaya, Papua, Indonesia

Authors: Tri Muji Ermayanti, Erwin Al Hafiizh, Ary Mandessy, Gesang Setyadi, Andi Mukhsia

Abstract

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Exploitation on plant population may put the endemic plants into an endangered state, hence, these plants will need to be conserved. In order to pursue conservation on endemic plants, we conducted in vitro seed germination and shoot multiplication of seven alpine and sub-alpine species endemic to Mount (Mt.) Jaya, in Papua, Indonesia, i.e. Tetramolopium klossii, Deschampsia klossii, Papuacalia cartenszensis, Epilobium hooglandii, Gaultheria novoguinensis, Rhododendron correoides and Rhododendron culminicolum. These species are categorized as slow-growth plants found in higher altitude (over 3700 m above sea level) and low temperature of Mt. Jaya. Seeds were surface-sterilized using Na-hypochloride and germinated aseptically on Murashige and Skoog (MS) medium. Dytikinin benzyl adenine (BA) was used for shoot multiplication. Seedling cultures were maintained in a controlled environment with continuous low light intensity (800 lux) and at temperature 26-27oC. Results showed that most species had more than 80% of germination rate on MS medium after a week in culture. BA was required to enhance shoots multiplication. Woody Plant (WP) (Lloyd & McCown, 1981) medium gave better shoot multiplication for R. culminicolum.