Tissue engineering and regenerative medicine are emerging fields focused on the development of tissues and organs that can be used in transplantation surgeries for the replacement or repair of damaged tissues or organs. This accomplishment would discard the need to use autologous and allogenic grafts, overcoming problems such as donor rejection, disease transmission or organ scarcity. The materials science field contributes directly for the achievement of this goal through the functionalization of natural or synthetic polymers or with the development of new biopolymers with diverse mechanical and biological characteristics. More recently, many research studies show the potential of using biotechnology approaches to generate new bioactive multifunctional materials engineered at the molecular level. An advantage of using genetically bioengineered biopolymers is the possibility of generating new genetic variants carrying different functional domains. The main goal of this thesis is to develop new multifunctional proteins that can be used in the fabrication of a new generation of biomaterials with improved features such as infection control, enhanced cell functions and better mechanical performance. As a result, the main objectives of this thesis are: I. Design of new functionalized spider silk fusion proteins through the combination of six repeats of the consensus unit for the native sequence of the major ampullate dragline silk I from Nephila clavipes (6mer) with two different types of proteins: bone sialoprotein (BSP) and the human antimicrobial peptides, namely human neutrophil defensin 2 (HNP‐2), human neutrophil defensins 4 (HNP‐4) and hepcidin. The four protein domains were fused with spider silk through recombinant DNA technology and the new chimeric proteins and silk alone were expressed in Escherichia coli RY‐3041 strain. 6mer, 6mer+BSP, 6mer+HNP‐2, 6mer+HNP‐4 and 6mer+hepcidin were purified with affinity chromatography and their identity was confirmed. II. Study the in vitro activity of the new chimeric proteins to verify if protein domains maintained their activity. Attenuated total reflection Fourier transform infrared (ATRFTIR) results show that the spider silk domain, 6mer, maintained its ability to selforganize into a β‐sheet conformation, an important feature related with its mechanical properties. Additionally, in vitro mineralization studies demonstrated that 6mer+BSP fusion protein with BSP retained the ability to induce the deposition of CaP. Finally, radial diffusion tests showed that the antimicrobial domains present in 6mer+HNP‐2, 6mer+HNP‐4 and 6mer+Hepcidin proteins maintained bactericidal activity. III. Assess the mechanical properties and roughness of 6mer+BSP protein films and its behaviour in the presence of calcium ions. Since 6mer+BSP was synthesized for bone regeneration applications the study of these parameters is important. In this way, atomic force microscopy (AFM) was used for imaging and for force spectroscopy. The results obtained show that 6mer+BSP had a higher stiffness than 6mer protein. For both proteins the roughness values were similar. In the presence of Ca ions, the AFM imaging showed the ability of 6mer+BSP chimeric protein to form supramolecular networks through ionic crosslinking. IV. Address the in vitro activity of the new chimeric proteins. Cell culture studies with human mesenchymal stem cells (hMSC) indicated that 6mer+BSP protein sustained hMSC proliferation and differentiation into the osteogenic lineage. In the case of 6mer+HNP‐2, 6mer+HNP‐4 and 6mer+hepcidin proteins in vitro cell studies demonstrated that these new proteins were capable of sustaining the proliferation of mammalian osteosarcoma cell line (SaOs‐2). V. Finally, based on the results obtained with the in vitro, 6mer+BSP and 6mer+hepcidin were selected for in vivo studies and the results showed no major differences between the inflammatory responses to the 6mer+BSP and 6mer+Hepcidin films and the responses observed for the controls 6mer, poly‐lactic‐glycolic‐acid (PLGA) and empty implants. The results obtained in this thesis demonstrated that 6mer+BSP, 6mer+HNP‐2, 6mer+HNP‐4 and 6mer+hepcidin bioengineered proteins represent promising proteinbased biomaterial for future biomedical applications. Additionally, the results reported here also highlight the potential of using new genetically engineered proteins to develop new hybrid multifunctional biomaterials for tissue engineering and regenerative medicine applications. This new generation of multifunctional proteins can combine different functionalities such as enhanced cell adhesion, mineral nucleation and infection control, and therefore exclude the need for additional chemical modification which has considerable disadvantages.