Map of DNA constructs. (A) The construct was designed to secrete the target protein human GLA (hGLA) under the cbh1 promoter (pcbh1) with the aid of a CBHI secretion signal (ss) and CBHI carrier fusion (cbh1), which was to be proteolytically cleaved at a KEX2 site (KEX) on route to secretion. A TEV protease site (TEV) was also included for downstream polishing after purification by Strep Tactin affinity using a strep2 tag (strep2) (B) Three variations of GLA were designed with RGD integrin (RGD) targeting for intracellular accumulation by ER retention of the Zera® peptide system (ZERA). The cbh1 locus was used as an integration site for all the constructs by homologous recombination using the cbh1 promoter sequence and a section of DNA downstream of the cbh1 region (cbh1 3’). The native terminator sequence (tcbh1) was followed by an Aspergillus nidulans AmdS (AmdS) marker gene for selective growth on acetamide. The size of the protein expressing region of the secretion construct was 2775 bp and 1632 bp for both RGD containing intracellular constructs whereas the control construct lacking RGD was 1563 bp.