In both examples, the hydrogen producing reactions are catalyzed by hydrogenases. Hydrogenases catalyze hydrogen activation by virtue of unique organometallic clusters that compose the catalytic sites. Based on the metal compositions of the catalytic sites, the hydrogenases organize into phylogenetic groups representing three Sasapyrine enzyme subclasses. The -hydrogenases, which are the focus of this investigation, generally exhibit a catalytic bias towards hydrogen production, possess high kcat values, monomeric compositions and show high sensitivity to oxygen. Because of these properties, -hydrogenases, as well as -hydrogenases, are being investigated to understand the structural basis for both fast hydrogen activation rates and for Homatropine Bromide oxygen tolerance towards achieving efficient natural and artificial solar hydrogen production systems. In recent years, several groups have developed different strategies for the recombinant expression of -hydrogenases. These include using either native hydrogenase expressing organisms as hosts, or Escherichia coli for heterologous expression. However, the maturation efficiencies based on the Fe content and specific activities indicated H-cluster incorporation was not complete. In addition, the purification of recombinant hydrogenases is most often performed by use of affinity tags, for example His or StrepII-tag, at the N- or C-terminus. In most cases affinity purification is performed as a single-step process that typically results in rather low yields, and purities at or greater than 80%. Although suitable for biochemical characterization, both crystallization and biophysical studies require production of large quantities of samples that are also high in purity and quality. Here, we show that the addition of the Fd as a fusion partner to HydA1 facilitated higher expression and purification yields of HydA1 hydrogenase. These effects were observed when Fd was fused to the N-terminus rather than the C-terminus of HydA1. Moreover, by incorporating a TEV protease site in the fusion linker it was possible to remove the Fd prior to the final affinity step, resulting in pure HydA1. The heterologous expression of metallo-enzymes, specifically hydrogenases, requires optimization of both protein expression and maturation processes, and each process can significantly affect the final production levels and enzyme qualities. In this study, we described an optimized protocol for recombinant production of -hydrogenase in E. coli developed from expression of an FdHydA1 fusion. We found that the major factors that contributed to increased expression levels were conditions that maintained more aerobic growth, and the utilization of Carbenicillin in place of Ampicillin at the start of anaerobic expression phase.