Refolding and simultaneous purification by three-phase partitioning of recombinant proteins from inclusion bodies.
Many recombinant eukaryotic proteins tend to form insoluble aggregates called inclusion bodies, especially when expressed in Escherichia coli. We report the first application of the technique of three-phase partitioning (TPP) to obtain correctly refolded active proteins from solubilized inclusion bodies. TPP was used for refolding 12 different proteins overexpressed in E. coli. In each case, the protein refolded by TPP gave either higher refolding yield than the earlier reported method or succeeded where earlier efforts have failed. TPP-refolded proteins were characterized and compared to conventionally purified proteins in terms of their spectral characteristics and/or biological activity. The methodology is scaleable and parallelizable and does not require subsequent concentration steps. This approach may serve as a useful complement to existing refolding strategies of diverse proteins from inclusion bodies.
Raghava
Smita
S
Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
Barua
Bipasha
B
Singh
Pradeep K
PK
Das
Mili
M
Madan
Lalima
L
Bhattacharyya
Sanchari
S
Bajaj
Kanika
K
Gopal
B
B
Varadarajan
Raghavan
R
Gupta
Munishwar N
MN
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015704:CD4 Antigens / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D017510:Protein Folding; D020673:Protein Renaturation; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000302:isolation & purification
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D015704:CD4 Antigens; C068356:CcdB protein, Plasmid F; D029721:Drosophila Proteins; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic; C490690:PTP-ER protein, Drosophila; D017027:Protein Tyrosine Phosphatases
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