Protein engineering (PE) is developing quickly in molecular biology, biochemistry, and computational science (Engqvist & Rabe, 2019). It involves designing and modifying proteins to enhance or decrease their activity, properties, and stability, or create new functions. Since proteins play a fundamental role in biology, understanding and manipulating protein activity and structure allow scientists to mimic their functions for diverse applications, including protein therapy, protein catalysts, and biosensor technologies (Lobzaev et al., 2024). The constant development of PE reflects this field dynamic and interdisciplinary nature, characterized by innovations in computational data, genome editing, and machine learning/ artificial intelligence (AI).

Currently, designing new proteins with desired functions is a complex process, but it highlights potential benefits to the biomedical science and industries (Gagner, Kim, & Chaikof, 2014). In general, the foundation of PE is based on a combination of protein structures and functions, which allows scientists to understand and change amino acid sequences with proposed applications (Watson et al., 2023). PE approaches have traditionally been divided into two principal strategies: directed evolution and rational design (Ovchinnikov & Huang, 2021) (Fig. 1). However, these methods have limitations in designing processes and data structures. Recently, semi-rational design has been highlighted as an attractive method to combine advances of these two traditional methods in protein applications (Chica, Doucet, & Pelletier, 2005). More proteins have been modified with the specific purposes of research to contribute to the dynamic growth of biology and drug development. However, about 10 % of proteins have known active binding sites that are indicated as drug targets for small molecules (Tabana et al., 2023). Therefore, the drugs indicated as small molecular-targeted therapy are limited to comparison with human diseases. These limitations motivate researchers to continue searching and developing more details in proteins.

To promote the development of proteins, recent years have reported remarkable technological advances that highlight the functions of proteins. The CRISPR/Cas system, an immune mechanism of a bacterium protecting against the invasion of foreign genetic elements and bacteriophages, has supported a wide range of PE with more accuracy (Xu & Li, 2020). Meanwhile, machine learning and AI have also made an evolution in controlling large datasets of protein sequences and structures to predict protein folding patterns and critical regions (Notin et al., 2024). These computational approaches complement experimental methods, which enhances the efficiency of protein workflows. Based on the development of PE, the protein application affects a wide range of fields, underscoring its transformative impact on society (Koths, 1995). In medicine, proteins have supported the foundation of innovative therapies such as antibodies, enzyme therapies, and next-generation vaccines, offering treatment options for patients with a higher quality of life (Koths, 1995). In the industrial sector, proteins have contributed to biocatalysis by reducing energy consumption and improving environmentally sustainable manufacturing processes (Guo et al., 2024). In addition, scientists can reconstruct novel biological systems with customized functions of targeted proteins in synthetic biology, which have a critical role in biosensors for detecting pathogens as well as for production in pharmaceuticals and fine chemicals.

Despite its remarkable achievements, PE faces several challenges that limit its application and innovation. The scalability of directed evolution experiments, the accuracy of computational biology, and the ethical implications of mimicking biological systems are critical areas that attract scientists with more research and attention (Hussain et al., 2017). In addition, ensuring the reproduction and reliability of proteins remains a significant hurdle in diverse contexts (Hussain et al., 2017). As the field advances, collaboration between biologists, chemists, and data scientists will play an essential role in overcoming these challenges and driving innovation, which will unlock the potential effects of novel proteins.

The advances of PE stand at the forefront of scientific and technological progress, offering potential opportunities to address complex challenges in medicines, industries, and sustainability. Through the updated results of the protein study, the primary purpose of this chapter is to overview the fundamental concepts, key techniques, and transformative applications of PE in research and industrial applications. The latest technological developments as well as future directions are described. Therefore, this chapter can provide updated technologies and applications in protein therapeutics, which hopefully will be helpful to researchers, students, as well as to any reader of a review paper.