AI Article Synopsis
- The production of recombinant proteins often faces challenges due to protein aggregation and inclusion body formation, leading to difficulties in obtaining functional proteins.
- This study introduces transverse urea gradient electrophoresis (TUGE) as a method to assess the folding capability of a pilin protein (TcpA-C) derived from El Tor, facilitating its recovery from inclusion bodies.
- The successful refolding and recovery of TcpA demonstrate its well-structured conformation, which holds potential for developing immunodiagnostics and cholera vaccines.
Article Abstract
The production of recombinant proteins in cells is often hampered by aggregation of newly synthesized proteins and formation of inclusion bodies. Here we propose the use of transverse urea gradient electrophoresis (TUGE) in testing the capability of folding of a recombinant protein from inclusion bodies dissolved in urea. A plasmid encoding the amino acid sequence 55-224 of TcpA pilin (C-terminal globular domain: TcpA-C) from El Tor enlarged by a His-tag on its N-terminus was expressed in cells. The major fraction (about 90%) of the target polypeptide was detected in cell debris. The polypeptide was isolated from the soluble fraction and recovered from inclusion bodies after their urea treatment. Some structural properties of the polypeptide from each sample proved identical. The refolding protocol was developed on the basis of TUGE data and successfully used for the protein large-scale recovery from inclusion bodies. Spectral, hydrodynamic, and thermodynamic characteristics of the recombinant TcpA recovered from inclusion bodies indicate the presence of a globular conformation with a pronounced secondary structure and a rigid tertiary structure, which is promising for the design of immunodiagnostics preparations aimed to assess the pilin level in different strains of and to develop cholera vaccines.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9695179 | PMC |
| http://dx.doi.org/10.3390/life12111802 | DOI Listing |
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