Recombinant biomaterials are they worth the trouble?

Abstract

Proteins play a crucial role in the field of biomaterials due to their inherent biocompatibility, bioactivity, and ability to interact with biological systems. As structural and functional components of natural tissues, proteins such as collagen, gelatin, fibrin, and silk fibroin are widely used in tissue engineering, drug delivery, and medical implants. However, the biological origin of these materials also raises questions about the risk of disease transfer or other ethical considerations. As a result, recombinant proteins are often proposed as a workaround. However, designing and expressing recombinant proteins is not straightforward and requires a good understanding of the necessary steps to translate a gene of interest into purified proteins that can be used as a biomaterial. The numerous interdependent experimental parameters make this field difficult to navigate for biomaterials scientists new to recombinant proteins. This contribution presents a systematic approach for the expression of elastin-like proteins (ELPs) that will help avoid common pitfalls and increase protein yield and purity. Plasmids encoding ELPs were generated based on a previously described method:[1] short oligonucleotide fragments were fused together and ligated into a pUC18 cloning vector, after which the full construct was transferred to a pET15b expression vector used to transform the typical expression strain E. coli BL21(DE). Expression conditions reported in literature were applied, followed by isolating the ELPs from the cell lysate using immobilised metal affinity chromatography (IMAC) or inverse transition cycling (ITC). Despite our best efforts, purification of the cell lysate initially resulted in minimal recovery of the protein of interest (PoI). To increase the expression yields alternative expression strains were considered together with adapting the experimental parameters, but this only resulted in minimal improvements. By starting from scratch and not relying on recommendations from literature, protein yields started to increase. By changing the sequence, the vector, replacing the expression strain and by optimising the expression parameters via a Design of Experiments approach, expression yields comparable to literature reports were finally obtained. The obtained results indicate that expression parameters depend on the primary sequence of the PoI, making it difficult to reproduce experimental results reported in literature and discouraging others from entering the field. However, many of these difficulties can be mitigated by applying a systematic approach tilting the balance in favour of the design freedom and unparallelled control that recombinant biomaterials offer.