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Sudjadi ., Sudjadi
Departement of Chemistry, Faculty of Pharmacy, University of Gadjah Mada
Medicine & Pharmacology

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THE OPTIMISED CONDITIONS OF INDUCTION OF RECOMBINANT RIP rMJC15310 ACTIVITY ISOLATED FROM Mirabilis jalapa L. LEAVES Astuti, Puji; ., Sudjadi; ., Sismindari
INDONESIAN JOURNAL OF PHARMACY Vol 23 No 2, 2012
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (836.5 KB) | DOI: 10.14499/indonesianjpharm0iss0pp93-98

Abstract

Ribosome Inactivating Proteins (RIPs) are compounds isolated from plants with ability to inhibit protein synthesis. The inhibition of protein synthesis is due to inactivation of ribosomal RNA through a site-specific  deadenylation  mediated  by  RNA  N-glycosidase. Reportedly, RIPs mainly possess wide range of bioactivity including antiviral  activity  against  plant  infections.  Other  activities  of  RIP were  as  abortifacien,  antivirus  and  anticancer.  This  study  was aimed  to  isolate  and  characterize  the  optimum  conditions  for inducing  the  expression  of  recombinant  RIPs  isolated  from  the leaves  of  Mirabilis  Jalapa  L.  We  have  been  successfully  isolated several  RIPs  and  engineered  these  proteins  to  be  expressed  in  E. coli. These recombinant proteins were obtained by screening cDNA library  originated  from  the  mRNA  of  Mirabilis  jalapa  L  leaves,  and inserted  into  pUC19  carrying  lacZ  gene.  The  presence  of recombinant  plasmid  was  tested  by  using  α-complementation assay. Many RIPs have been isolated from plants and these proteins express  enzymatic  activity  by  cutting  supercoiled  double  stranded DNA. One RIP namely rMJC15310 was obtained from this study and the  proteins  having  ~  8kb  in  size,  cut  the  supercoiled  DNA  into linear  form  at  the  concentration  as  low  as  5  µg.  The  ability  to  cut supercoiled  DNA  increased  on  inducing  its  expression  with  0.4% IPTG.Key words:   Ribosome  Inactivating  Proteins  (RIP),  IPTG,  Mirabilis  jalapa L., recombinant protein 
Effects of pH, temperature and storage on the stability of MJ-30 protein isolated from Mirabilis jalapa L leaves ., Sudjadi; Ikawati, Zulies; ., Sismindari; Rahayu, Putu Riana Suastari
INDONESIAN JOURNAL OF PHARMACY Vol 15 No 1, 2004
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (358.748 KB) | DOI: 10.14499/indonesianjpharm0iss0pp1-6

Abstract

The total protein of Mirabilis jalapa L leaves had the ability to cleave supercoiled DNA and showed toxicity on HeLa and Raji cell-lines. The 30 kD protein (MJ-30) purified by cationic exchange chromatography possessed activites as RIP e.g DNA supercoiled cleavage and RNA N-glycosidase activity. The aim of this study was to prepare pure MJ-30 and to observe the stability of MJ-30 .MJ-30 of M.jalapa L leaves was purified using the combination of ammonium sulphate fractionation and cationic exchange chromatography with NaCl gradient elution. MJ-30 was subjected for its stability to assays against pH, temperature, and storage period.Stability assay showed that MJ-30 is stabil at pH 5-6, then the activity decreased as the pH increased. This protein was stable at 300 – 550C, and the activity decreased when the temperature increased. Storage at 40C, MJ-30 was stable until 12 days but the activity was decreasing. However, at 300C, MJ-30 was stable for 3 days only. Glycerol addition to the MJ-30 solution has made the activity stable for 18 days at 40C and 300C storage.Keyword : Protein MJ-30, Leaves of Mirabilis jalapa L, stability, pH, temperature, storage
Co-Authors Puji Astuti Putu Riana Suastari Rahayu, Putu Riana Suastari Sismindari . Zulies Ikawati, Zulies