Krishna Mohan Padmanabha Das

Publications (4) View all

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  Available from: Krishna Mohan Padmanabha Das

  Article: Cloning ,expression of soluble hGMCSf and purification by onestep affinity chromatography

  Krishna Mohan Padmanabha Das, Sampali Banerjee, Sandeep Somani, rahul Nair, Nivedita shekhar, shweta jain, karpagavalli, Sriram Padmanabhan
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  ABSTRACT: Abstract: Expression of human granulocyte macrophage colony stimulating factor (hGMCSF), a cytokine of therapeutic importance, as a thioredoxin (TRX) fusion has been investigated in Escherichia coli BL21 (DE3) codon plus cells. The expression of this protein was low when cloned under the T7 promoter without any fusion tags. High yield of GMCSF was achieved (~88 mg/L of fermentation broth) in the shake flask when the gene was fused to the E. coli TRX gene. The protein was purified using a single step Ni2+-NTA affinity chromatography and the column bound fusion tag was removed by on-column cleavage with enterokinase. The recombinant hGMCSF was expressed as a soluble and OPEN ACCESS Int. J. Mol. Sci. 2011, 12 2065 biologically active protein in E. coli, and upon purification, the final yield was ~44 mg/L in shake flask with a specific activity of 2.3 × 108 U/mg. The results of Western blot and RP-HPLC analyses, along with biological activity using the TF-1 cell line, established the identity of the purified hGMCSF. In this paper, we report the highest yield of hGMCSF expressed in E. coli. The bioreactor study shows that the yield of hGMCSF could be easily scalable with a yield of ~400 mg/L, opening up new opportunities for large scale production hGMCSF in E. coli.
  international Journal of Molecular Sciences. 01/2011;
• Article: An alternate method for purification of recombinant Taq DNA polymerase using aqeuos two phase system

  Krishna Mohan Padmanabha Das, Sampali Banerjee, Sriram Padmanabhan
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  ABSTRACT: A simple and cost-efficient method for purification of E. coli derived recombinant Taq DNA polymerase isolated from the thermophilic bacterium, Thermus aquaticus, is described. The recombinant Taq DNA polymerase was purified using an aqueous two-phase extraction method following a primary treatment of heat denaturation. The extraction method selectively enriched Taq DNA polymerase in the lower salt phase with complete recovery and high specific activity. Such a purified Taq DNA polymerase protein displayed nearly 8 fold purification with ~ 117 % activity recovery with insignificant amount of host DNA. This process was found to be rapid and scalable in comparison to the conventional ammonium sulfate precipitation method.
  Industrial Biotechnology. 01/2010;
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  Article: A Novel Thermo stability Conferring Property of Cherry Tag and its Application in Purification of Fusion Proteins

  Krishna Mohan Padmanabha Das, Shruti Barve, Sampali Banerjee, Suman Bandyopadhyay, Sriram Padmanabhan
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  ABSTRACT: Cherry tag, a red polypeptide of the heme binding part of cytochrome is used to attain high levels of soluble protein expression in E. coli. A novel heat stability conferring property of this tag was observed and studied for constructs of two soluble fusions especially Cherry-Granulocyte colony stimulating factor (GCSF) and Cherry- Staphylokinase (SAK). Heat incubation of these fusion proteins at 70oC for 20 minutes culminated in specific denaturation and precipitation of E. coli proteins excluding the fusion proteins. Both the heat treated fusion proteins were found to be functionally active. Thus Cherry fusion tag could be used as a cost-efficient tool in purification of proteins by imparting heat stability.
  Journal of Microbial and Biochemical technology. 01/2010; 1:1: 059-063,2010.
• Source

  Available from: Sriram Padmanabhan

  Article: A Novel Thermostability Conferring Property of Cherry Tag and its Application in Purification of Fusion Proteins

  Krishna Mohan Padmanabha Das, Barve Shruti, Banerjee Sampali, Bandyopadhyay Suman, Padmanabhan Sriram
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  ABSTRACT: Cherry tag, a red polypeptide of the heme binding partof cytochrome is used to attain high levels of soluble proteinexpression in E. coli. A novel heat stability conferringproperty of this tag was observed and studied for constructsof two soluble fusions especially Cherry-Granulocytecolony stimulating factor (GCSF) and Cherry-Staphylokinase (SAK). Heat incubation of these fusionproteins at 70oC for 20 minutes culminated in specificdenaturation and precipitation of E. coli proteins excludingthe fusion proteins. Both the heat treated fusion proteinswere found to be functionally active. Thus Cherryfusion tag could be used as a cost-efficient tool in purificationof proteins by imparting heat stability.
  Journal of Microbial & Biochemical Technology. 01/2009;