Many eukaryotic proteins are attached to the cell membrane via Glycosylphosphatidyl-inositol (GPI) anchors that are added post-translationally to the C-terminus of proteins. These complex structures contain a highly conserved carbohydrate core, variable number of phosphate residues and lipid chains. The functions of GPI-anchored proteins (GPI-APs) are widespread, including participation in signal transduction, immune response regulation, lipid raft partitioning and prion disease pathogenesis. However, the effect of the GPI-anchor itself in these processes still remains unclear. Due to their high complexity and metabolic expense, a function beyond membrane anchoring has been anticipated. GPI-APs are not accessible in a homogeneous form and high amounts by isolation from natural sources, making investigation of the GPI effects on the function and structure of the protein difficult. Some progress has been made in the field of total GPI synthesis and small GPI-anchored peptides are now accessible by chemical synthesis, but these strategies are limited regarding peptide length by the scope of solid-phase peptide synthesis (SPPS). So far, no method is available for the routine generation of homogeneous GPI-APs for structural or biological studies. In this work, different semi-synthetic, intein-based methods have been investigated and established for the generation of homogeneous GPI-anchored proteins, Figure 1. The first method, Expressed Protein Ligation (EPL, a), utilizes the GyraseA intein from Mycobacterium xenopi for the generation of protein α-thioesters which are then ligated to cysteine-containing GPI-anchors. The second method, Protein Trans-Splicing (PTS, b) uses the naturally split DnaE intein from Nostoc punctiforme. For PTS, proteins of interest are expressed in E. coli as fusion proteins with the larger N-terminal split intein fragment NpuN, whereas the shorter (39 amino acids) C-terminal fragment NpuC is synthesized by SPPS and ligated to a cysteine-containing GPI-anchor. Subsequently both fragments are combined and trans-splicing takes place initiated by association of the fragments and folding into a full intein structure, ligating both exteins together with a native peptide bond. This method requires the introduction of some extra amino acid residues which are essential for the splicing reaction. Both EPL and PTS were successfully established for the anchoring of eGFP as a model protein to biotin and a GPI anchor containing the full pseudo-pentasaccharide core structure, phosphates, and a simplified monolipid chain. Application of these strategies to the semi-synthesis of the naturally GPI-anchored proteins Thy-1 and prion protein (PrP) showed, however, that thioester generation is inefficient under denaturing conditions, making the EPL strategy less useful for our purpose. PTS on the other hand was efficient under denaturing conditions, although slower. In comparison to the natural conditions, this strategy suffered from insufficient availability of the synthetic GPI-coupled peptide due to a high synthetic effort required for its synthesis. Therefore, a third semi-synthetic, intein-based method was developed: One-Pot-Ligation (OPL, c). In OPL, the same fusion proteins required for PTS can be used, but they are combined with a mutated C-terminal fragment, NpuC(AA), in which two essential amino acid residues are exchanged for alanines, rendering the C-terminus of the split intein function-less. Instead, with this system, a protein intermediate can be captured and protein thioesters can be generated in situ by the additions of external thiol reagents, with subsequent ligation of Cys-GPI in a one-pot manner. This strategy was successfully applied for anchoring both soluble and denatured proteins (eGFP, Thy-1, PrP and IL-2) to biotin, dimannose and mono- and bilipidated GPI-anchors. It is more versatile towards the generation of GPI-AP libraries, however exhibited some immature protein thioester hydrolysis which could largely be overcome by the use of more stable thiols. Additional challenges arose from characterization of these highly complex protein-carbohydrate-lipid conjugates, carrying multiple charges, in LC-MS. Although progress could be made towards this goal, a complete characterization was not achieved for each protein. Initial structural characterization of the obtained GPI-APs was performed for eGFP-GPI using circular dichroism (CD). This study showed that using any of the three methods investigated, anchoring to GPIs does not affect the structure of eGFP.