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( Rameshkumar ),( Neelamegam ),( Niraikulam Ayyadurai ),( Nagarajan Kayalvizhi ),( Paramasamy Gunasekaran ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.1
The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.
Edwardraja, Selvakumar,Neelamegam, Rameshkumar,Ramadoss, Vijayaraj,Venkatesan, Subramanian,Lee, Sun-Gu Wiley Subscription Services, Inc., A Wiley Company 2010 Biotechnology and bioengineering Vol.106 No.3
<P>Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti-c-Met scFv, which was a cytoplasmic non-foldable protein, was redesigned as the model system. This study confirmed that the engineered anti-c-Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c-Met antigen. The structures of the wild-type anti-c-Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition. Biotechnol. Bioeng. 2010; 106: 367–375. © 2010 Wiley Periodicals, Inc.</P>
Niraikulam Ayyadurai,Rameshkumar Neelamegam,Soundrarajan Nagasundarapandian,Selvakumar Edwardraja,Hyung Soon Park,Soo Jae Lee,Tae Hyeon Yoo,Hyungdon Yoon,이선구 한국생물공학회 2009 Biotechnology and Bioprocess Engineering Vol.14 No.3
In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while Lhomopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/ MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while Lhomopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/ MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system
Selvakumar, Edwardraja,Rameshkumar, Neelamegam,Lee, Sun-Gu,Lee, Soo-Jae,Park, Hyung-Soon WILEY-VCH Verlag 2010 Chembiochem Vol.11 No.4
<B>Graphic Abstract</B> <P>Just where you want it: Grafting target scFv complementarity-determining regions onto stable frameworks (FR) and consensus-based engineering of methionines on the FR enables the production of methionine-free scFv that can be specifically modified with a bio-orthogonal reactive group on its N-terminal through in vivo incorporation of a methionine analogue. <img src='wiley_img_2010/14394227-2010-11-4-CBIC200900685-content.gif' alt='wiley_img_2010/14394227-2010-11-4-CBIC200900685-content'> </P>
The Effect of the cspA 5'-Untranslated Region on Recombinant Protein Production at Low Temperature
Su-Hyun Kim,허미애,김소연,Yu-Jin Kim,Rameshkumar Neelamegam,이선구 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.3
Expression of recombinant proteins in Escherichia coli is often carried out at low temperatures so as to improve the solubility and stability of the expressed proteins. However, the use of low temperature significantly limits the growth rate of the expression host, leading to a reduction in protein productivity. Here, we show that the introduction of the cspA 5'-untranslated region (cspA 5'-UTR) onto the transcript of a target gene enhances the expression levels via the T7 promoter at low temperatures. First, the effects of the cspA 5'-UTR sequence and CspA coexpression on green fluorescence protein (GFP) production were investigated. While the cspA 5'-UTR sequence had no influence on the expression rate of GFP at 37℃, it improved the expression level of GFP by approximately 1.5 fold at 15℃. Coexpression of the CspA protein further increased the expression level of GFP only slightly in the presence of the cspA 5'-UTR. Subsequently, the effect of the cspA 5'-UTR on protein production was examined for six different proteins (CysS, HisS, ProS, ThrS, TrpS, and YadB) at 15℃. The introduction of cspA 5'-UTR sequence led to approximate two-fold improvement for HisS and ThrS production, a slight improvement for TrpS and YadB, and no influence on CysS and ProS protein production. Therefore, the effect of the cspA 5'-UTR on the expression level at low temperature varied depending on the target gene.