| dc.description.abstract | The induction of metallothionein (MT) expression in mitochondrial disorders has been well studied on the transcription level by means of RNA measurements in an attempt to understand and confirm the function of this protein in the deficient cells and organs (Olivier, 2004:42; Pretorius, 2006:44; Reinecke, 2004:89). However, MT expression induction still needs to be verified on protein (translation) level in order to confirm previous findings and to gain better perspective on the significance of MT expression induction. Therefore, it is necessary to use a technique that is capable of quantifying MT accurately in biological material. Due to the lack of sensitivity and selectivity of many commonly used techniques (Dabrio et al., 2002:125), it is necessary to develop a mass spectrometric based quantification technique to detect and quantify MT-2A selectively and accurately.
For quantification of human MT-2A in biological material using a mass spectrometry-based method, a MT (MT-2A) standard similar to the native form but with a slightly different mass was required. Due to the lack of pure human MT standards and high cost of pure rabbit MT standards, it was decided to create a recombinant human MT-2A with different mass due to additional N-terminal amino acids. In addition, native human MT-2A is also required to develop and optimize an MS quantification technique in a future study. Therefore, pure (98 %) rabbit MT standard, which is highly similar to human MT-2A, was purchased to serve as a positive control for MS detection in this study and which can also be used to develop and optimize an MS quantification technique in a future study. An expression vector for human MT-2A was constructed with the use of recombinant DNA techniques. The correct construct was identified and characterized with PCR and verified by sequencing. This newly created expression vector was transformed into four E.coli BL21(DE3) strains to express a modified human recombinant MT-2A (MT-2AA) using induction with IPTG. This protein comprised of a full length human MT-2A sequence, but excluding the N-terminal Met and including an N-terminal His-tag. MT-2AA expression in the selected strains was extensively optimized and monitored with SDS-PAGE. Ecoli BL21 (DE3) CodonPlus-RIL cells proved to be the strain that expressed MT-2'A at the highest relative levels. Expressed MT-2'A was isolated and purified using a three step purification procedure which included heat treatment, metal chelating chromatography and RP-HPLC. Relative pure (70 %) MT-2'A was successfully obtained as confirmed with SDS-PAGE and mass spectrometry. Removal of the His-tag from MT-2'A with thrombin protease cleavage was, however, unsuccessful. In addition, it was observed that this protein was, compared to native commercially obtained MT-2A, unstable and after extensive purification still had a lower than required purity. It was concluded from this studies' results that, although it was successfully produced, this recombinant MT-2A protein would not be suitable as an internal standard for MS analysis of human MT-2A. On the other hand, rabbit MT-2E (as alternative) holds great promise as internal standard since it is stable and pure. | |
