To circumvent the need for in vitro processing, we included a synthetic furin recognition motif between the protein domains of MBP-GrB fusion proteins and investigated potential in vivo cleavage by endogenous furin-like proteases from yeast. Analysis of P. pastoris culture supernatants revealed that fusion to MBP markedly enhanced the amount of soluble GrB, while this was not the case when GrB was fused to GST. GrB was liberated from MBP by specific in vivo cleavage during secretion, allowing direct isolation of the enzyme in processed form. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions, indicating that this approach can generally be applied to enhance secretion and yields of functionally active recombinant proteins. To investigate the fate of GST and MBP domains after expression of the fusion proteins. Thereby no unprocessed GST-furS-GrB or MBP-furS-GrB proteins were detected, confirming the results with GrB-specific antibody shown above. However, we observed an MG132 increase over time of soluble GST and MBP, that reflected the previously observed levels of processed GrB. The apparent molecular weights of the single bands detected correspond well with the calculated molecular weights of the MBP and GST moieties. This indicates complete processing of the fusion proteins by cleavage within the furS recognition site in the secretory pathway, resulting in secretion of processed GrB and both fusion partners GST and MBP. Proteins that are secreted by the exocytosis pathway translocate into the membrane or lumen of the endoplasmic reticulum, and are then transported to the Golgi apparatus for sorting to cellular destinations. Misfolded proteins are retained in the ER, and in some cases are retranslocated from the ER and subjected to ER-associated degradation by the ubiquitin-proteasome system. To investigate potential intracellular retention of the GrB fusion proteins, yeast cell lysates were prepared and analyzed with GrB-specific antibody. For unfused GrB included for comparison we detected two bands in the intracellular fraction migrating at approximately 44 and 53 kDa, apparently corresponding to processed glycosylated GrB and glycosylated GrB still containing the signal peptide. In contrast, only marginal amounts of unprocessed GST-furS-GrB and MBP-furS-GrB fusion proteins were found, together with two additional smaller bands likely representing MBP-furS-GrB degradation products. These data show that a large proportion of unfused GrB was retained in an intracellular compartment likely representing the ER, while GST and MBP domains both promoted secretion of GrB. However, only MBP was able to enhance the overall level of GrB. Expression of recombinant proteins.