Protein microarray have become an important tool for advancing disease diagnostics and proteomics analysis due to their ability in providing high throughput screening. Since screening occurred in a dedicated region on an array surface, the ability to spatially pattern protein in a simple and general manner is essential. In this work, we report a new method to reversibly immobilize proteins to a surface in a functionally active orientation directly from cell lysate by employing a fusion protein consists of a thermal-responsive elastin domain as the surface anchor and a calcium-responsive calmodulin domain for protein capturing. Incorporation of a M13 tag into recombinant proteins enables not only easy surface immobilization but also direct purification from cell lysates. The feasibility of concept was demonstrated using the M13-tagged yellow fluorescent protein (M13TYFP). The proper orientation of calmodulin domain was achieved by selective adhesion of elastin domain onto a hydrophobic surface. The resulting functionalized surfaces were shown to capture M13TYFP directly from cell lysate through the specific calmodulin-M13 association in a calcium dependent manner. We also demonstrated that immobilization is reversible; the bound proteins were released from the surface in the presence of calcium chelators.