Characterization of a human inhibitory antibody fragment against tissue factor

[摘要] English: Tissue factor is a transmembrane glycoprotein that functions as the primary initiator of coagulation in response to mechanical or chemical damage. Due to its key position within the coagulation cascade it also plays an important role in the pathology of thrombosis and thrombotic complications associated with cardiovascular disease as well as in non-thrombotic disorders and diseases such as obesity, diabetes, cancer and HIV-AIDS. Recognising the potential in tissue factor inhibition as a novel approach to antithrombotic therapy, our laboratory utilized phage display technology in a previous study, in order to identify a 26 kDa single chain antibody fragment which functionally inhibits human tissue factor. In the current study, the tissue factor inhibitory single chain variable fragment (TFIscFv) was expressed by means of the pIT2 plasmid vector by Escherichia coli HB2151. This expression system was utilised in an up-scale setting in an attempt to improve the TFI-scFv yield. Although functional TFI-scFv was successfully purified from the culture media by means of Protein A affinity chromatography, the process was hampered by large sample volumes, low levels of expression as well as the high cost involved in Protein A purification. Due to an initial focus on improving TFI-scFv yield through the processing of larger sample volumes rather than the improvement of the expression system, immobilised nickel affinity chromatography was investigated as a more cost effective alternative to Protein A affinity chromatography. It was found that the original expression system was incompatible with immobilised nickel affinity chromatography as the protein was expressed into the culture media. The culture media contained nickel chelating elements that stripped the nickel from the column and consequently prevented TFIscFv purification. Subsequently the TFI-scFv gene was isolated, cloned into an over-expression system and modified to redirect the expression to the bacterial cytoplasm. Although TFI-scFv was successfully redirected to the bacterial cytoplasm and purified by means of nickel affinity chromatography, it was found that expression was hampered due to the presence of rare codons. The detrimental effect of rare codons on TFI scFv yield was addressed through the modification of the expression host by the coexpression of the pRARE plasmid as well as by the rare codon optimization of the TFI-scFv gene sequence for expression in E. coli. Although the co-expression of the pRARE plasmid only slightly improved TFI-scFv yield, a sufficient amount of TFIscFv was generated for functional testing. The modified TFI-scFv displayed a similar inhibition effect with reference to the original construct. The rare codon optimisation resulted in a substantial increase in TFI-scFv yield but consequently resulted in the loss of solubility and the production of inclusion bodies. Although the loss of TFIscFv solubility is unwanted, the high level of expression achieved provides an ideal platform for the further development and characterization TFI-scFv in animal thrombosis models.

[发布机构] University of the Free State