We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification.
Human alpha-fetoprotein (hAFP) is the main human oncofetal protein. Receptor of hAFP is expressed on the surface of different types of cancer cells, but not produced by normal cells of the adult organism. The hAFP interacts with the receptor via its third domain. The conjugates of native hAFP with a variety of natural cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The C-terminal hAFP fragment comprising amino acids from 404 to 595 of the native hAFP was expressed in E. coli BL21 (DE3) strain. The level of the protein expression was no less than 150 mg/l. Highly efficient purification and refolding procedures were developed. The final protein yield was no less than 50% with purity of about 95%. Refolded rAFP3D bound specifically with cancer cells carrying hAFP receptor and was accumulated by them. This rAFP3D can be used as a carrier for the targeted drug delivery to cancer cells.
Hydrophobic recombinant proteins often tend to aggregate upon expression into inclusion bodies and are difficult to refold. Producing them in soluble forms constitutes a common bottleneck problem. A fusion system for production of insoluble hydrophobic proteins in soluble stable forms with thermophilic minichaperone, GroEL apical domain (GrAD) as a carrier, has recently been developed. To provide the utmost flexibility of the system for interactions between the carrier and various target protein moieties a strategy of making permutated protein variants by gene engineering has been applied: the original N- and C-termini of the minichaperone were linked together by a polypeptide linker and new N- and C-termini were made at desired parts of the protein surface. Two permutated GrAD forms were created and analyzed. Constructs of GrAD and both of its permutated forms fused with the initially insoluble N-terminal fragment of hepatitis C virus’ E2 protein were tested. Expressed fusions formed inclusion bodies. After denaturation, all fusions were completely renatured in stable soluble forms. A variety of permutated GrAD variants can be created. The versatile format of the system provides opportunities for choosing an optimal pair between particular target protein moiety and the best-suited original or specific permutated carrier.
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