title: Mycobacterium tuberculosis H37Ra: a surrogate for the expression of conserved, multimeric proteins of M.tb H37Rv.
abstract: BACKGROUND
  Obtaining sufficient quantities of recombinant M.tb proteins using traditional approaches is often unsuccessful. Several enzymes of the glycolytic cycle are known to be multifunctional, however relatively few enzymes from M.tb H37Rv have been characterized in the context of their enzymatic and pleiotropic roles. One of the primary reasons is the difficulty in obtaining sufficient amounts of functionally active protein.
  
  
  RESULTS
  In the current study, using M.tb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) we demonstrate that expression in E. coli or M. smegmatis results in insolubility and improper subcellular localization. In addition, expression of such conserved multisubunit proteins poses the problem of heteromerization with host homologues. Importantly the expression host dramatically affected the yield and functionality of GAPDH in terms of both enzymatic activity and moonlighting function (transferrin binding). The applicability of this system was further confirmed using two additional enzymes i.e. M.tb Pyruvate kinase and Enolase.
  
  
  CONCLUSIONS
  Our studies establish that the attenuated strain M.tb H37Ra is a suitable host for the expression of highly hydrophobic, conserved, multimeric proteins of M.tb H37Rv. Significantly, this expression host overcomes the limitations of E. coli and M. smegmatis expression and yields recombinant protein that is qualitatively superior to that obtained by traditional methods. The current study highlights the fact that protein functionality (which is an an essential requirement for all in vitro assays and drug development) may be altered by the choice of expression host.
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
authors: Vishant Mahendra
authors: VM
authors: Boradia
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
authors: Pravinkumar
authors: P
authors: Patil
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
authors: Anushri
authors: A
authors: Agnihotri
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
authors: Ajay
authors: A
authors: Kumar
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
authors: Kalpesh Kumar
authors: KK
authors: Rajwadi
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
authors: Ankit
authors: A
authors: Sahu
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
authors: Naveen
authors: N
authors: Bhagath
authors: Council of Scientific and Industrial Research-Institute of Microbial Technology (CSIR-IMTECH), Sector 39 A, Chandigarh, 160036, India.
authors: Navdeep
authors: N
authors: Sheokand
authors: Council of Scientific and Industrial Research-Institute of Microbial Technology (CSIR-IMTECH), Sector 39 A, Chandigarh, 160036, India.
authors: Manoj
authors: M
authors: Kumar
authors: Council of Scientific and Industrial Research-Institute of Microbial Technology (CSIR-IMTECH), Sector 39 A, Chandigarh, 160036, India.
authors: Himanshu
authors: H
authors: Malhotra
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
authors: Rachita
authors: R
authors: Patkar
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India.
authors: Navi
authors: N
authors: Hasan
authors: Council of Scientific and Industrial Research-Institute of Microbial Technology (CSIR-IMTECH), Sector 39 A, Chandigarh, 160036, India.
authors: Manoj
authors: M
authors: Raje
authors: National Institute of Pharmaceutical Education and Research (NIPER), Phase X, Sector 67, SAS Nagar, Punjab, 160062, India. chaaya@niper.ac.in.|Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Phase X, SAS Nagar, Punjab, 160062, India. chaaya@niper.ac.in.
authors: Chaaya Iyengar
authors: CI
authors: Raje
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D010751:Phosphopyruvate Hydratase / Q000235:genetics; D055503:Protein Multimerization; D011770:Pyruvate Kinase / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D014168:Transferrin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D014168:Transferrin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011770:Pyruvate Kinase; D010751:Phosphopyruvate Hydratase