ABSTRACT Certain recombinant proteins are deemed “difficult to express” in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP‐2 and TIMP‐3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP‐2 and TIMP‐3 are detectable at mRNA and protein level within the cell but only TIMP‐2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP‐3 is severely limited in its post‐translational processing. To overcome this challenge, modification of the TIMP‐3 sequence to include a furin protease‐cleavable pro‐sequence resulted in secretion of the modified TIMP‐3 protein, however, incomplete processing was observed. Based on the TIMP‐3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP‐4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP‐4) and non‐secreted (TIMP‐3) targets, and presents a novel strategy to enhance the production of “difficult” recombinant targets. Biotechnol. Bioeng. 2017;114: 2348–2359. © 2017 Wiley Periodicals, Inc. In this study, the authors present a systematic screen to characterise the mechanisms that discriminate between secretion of recombinant proteins with extensive sequence homology. A protein engineering approach was employed enhanced the production of ‘difficult to express’ proteins from the tissue inhibitor of metalloproteinase (TIMP) family. The addition of a furin‐cleavable pro‐sequence resulted in successful secretion of non‐secreted (TIMP‐3) and poorly secreted (TIMP‐4) targets and presents a novel strategy to increase the production of “difficult” recombinant targets.