Construction And Application Of An Intein-medicated Prokaryotic Expression And Purification Vector

Protein splicing is a newly discovered posttranslational editing process thatremoves an internal protein fragment from the protein precursor. During the splicingprocess the internal protein fragment, intein, triggered the self-excision from theprecursor protein and the concomitant ligation of the flanking protein fragments,exteins. The self-catalyses requires neither auxiliary enzymes nor cofactors and onlyinvolves four intramolecular reactions. In vector pTWIN, chitin binding domain fused at intein's N-terminal can bind tothe chitin resin. When target protein is fused at intein's C-terminal, at pH=7 and 25intein is induced to self-cleavage between itself and target protein and then the targetprotein is released. The purification system based on intein self-cleavage is a singlechromatographic step which don't need to use protein enzyme and so avoid thenon-specific degradation and further removal of the protein enzyme. When gene engineering antibody is expressed in large yield in E.coli, it is oftenprone to be present in the form of insoluble inclusion body. The main reason for thatis gene engineering antibody is a reconstructed molecule on the basis of naturalantibody and can only be folded correctly with an aid of foldases and chaperones. Butthe number of foldases and chaperones constitutively expressed is not enough tofacilitate the correct fold of novel proteins and so the insoluble products are formed.There is cis- or trans-form of peptide bond linked by residue Proline and thetrans-form is predominant in natural proteins. Peptidyl-prolyl cis, trans-isomerasesare required for catalyzing the isomerization of cis-bond to trans-bond, one ofrate-limited steps of protein folding. FkpA is one of FKBP protein family and E. coliperiplasmic heat shock proteins which has two kinds of functions,namelycis,tran-isomerase and chaperone. Definitely speaking, FkpA can prevent the earlyintermediates from aggregation by binding to it and so can effectively improve theamount of soluble target proteins. In order to enhance the ratio of soluble proteins, FkpA is used to fusion-expressedand coexpressed with the target protein. The result show that scFv can bind to chitinhigh efficiently and self-cleavage of intein is also very high. ScFv is only present inthe SDS elution fraction. When coexpressed with FkpA, scFv has appreciately highersolubility,but don't have any improvement of solubility fused with FkpA.