Overexpression, on-column refolding and isotopic labeling of Hahellin from Hahella chejuensis, a putative member of the betagamma-crystallin superfamily.
A gene which encodes a hypothetical protein of 40 kDa has been identified in the genome of a marine bacterium Hahella chejuensis, as a putative member of betagamma-crystallin superfamily. This hypothetical protein contains a putative betagamma-crystallin-like domain, along with other domains for carbohydrate binding regions. It is named as Hahellin. A PCR amplified stretch of 92-amino acid residue long protein was cloned into pET21a vector and overexpressed in Escherichia coli strain BL21(DE3)pLysS cells. The recombinant Hahellin, produced as inclusion bodies, was estimated to be around 50% of the total cellular protein content which was solubilized in 8 M urea. The protein was purified and refolded using an anion exchange column. The MALDI-TOF mass spectrometry revealed the purity and monomeric nature of the protein. Further, a method to prepare isotopically (15N/13C) labeled protein with high yield for NMR studies is reported. The uniformly 15N-labeled Hahellin thus produced has been characterized by recording a sensitivity enhanced 2D [15N]-[1H] HSQC spectrum. The well, dispersed peaks in the spectrum confirm that the protein is indeed well folded and suitable for further studies by NMR.
Srivastava
Atul K
AK
Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai 400005, India.
Sharma
Yogendra
Y
Chary
Kandala V R
KV
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D002247:Carbon Isotopes; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003459:Crystallins / Q000096:biosynthesis* / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D020563:Gammaproteobacteria / Q000737:chemistry / Q000235:genetics*; D002479:Inclusion Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D003459:Crystallins; D009587:Nitrogen Isotopes