Current Protein and Peptide Science - Volume 20, Issue 5, 2019

Proteins are one of the most important and resourceful biomolecules that find applications in health, industry, medicine, research, and biotechnology. Given its tremendous relevance, protein engineering has emerged as significant biotechnological intervention in this area. Strategic utilization of protein engineering methods and approaches has enabled better enzymatic properties, better stability, increased catalytic activity and most importantly, interesting and wide range applicability of proteins. In fact, the commercialization of engineered proteins have manifested in economically beneficial and viable solutions for industry and healthcare sector. Protein engineering has also evolved to become a powerful tool contributing significantly to the developments in both synthetic biology and metabolic engineering. The present review revisits the current trends in protein engineering approaches such as rational design, directed evolution, de novo design, computational approaches etc. and encompasses the recent progresses made in this field over the last few years. The review also throws light on advanced or futuristic protein engineering aspects, which are being explored for design and development of novel proteins with improved properties or advanced applications.

Protein splicing domains, also called inteins, have become a powerful biotechnological tool for applications involving molecular biology and protein engineering. Early applications of inteins focused on self-cleaving affinity tags, generation of recombinant polypeptide α-thioesters for the production of semisynthetic proteins and backbone cyclized polypeptides. The discovery of naturallyoccurring split-inteins has allowed the development of novel approaches for the selective modification of proteins both in vitro and in vivo. This review gives a general introduction to protein splicing with a focus on their role in expanding the applications of intein-based technologies in protein engineering and chemical biology.

The fluorinated alcohol 2,2,2-Trifluoroethanol (TFE) has been implemented for many decades now in conformational studies of proteins and peptides. In peptides, which are often disordered in aqueous solutions, TFE acts as secondary structure stabilizer and primarily induces an α -helical conformation. The exact mechanism through which TFE plays its stabilizing roles is still debated and direct and indirect routes, relying either on straight interaction between TFE and molecules or indirect pathways based on perturbation of solvation sphere, have been proposed. Another still unanswered question is the capacity of TFE to favor in peptides a bioactive or a native-like conformation rather than simply stimulate the raise of secondary structure elements that reflect only the inherent propensity of a specific amino-acid sequence. In protein studies, TFE destroys unique protein tertiary structure and often leads to the formation of non-native secondary structure elements, but, interestingly, gives some hints about early folding intermediates. In this review, we will summarize proposed mechanisms of TFE actions. We will also describe several examples, in which TFE has been successfully used to reveal structural properties of different molecular systems, including antimicrobial and aggregation-prone peptides, as well as globular folded and intrinsically disordered proteins.

L-asparaginase is a valuable protein therapeutic drug utilized for the treatment of leukemia and lymphomas. Administration of asparaginase leads to asparagine starvation causing inhibition of protein synthesis, growth, and proliferation of tumor cells. Besides its clinical significance, the enzyme also finds application in the food sector for mitigation of a cancer-causing agent acrylamide. The numerous applications ensue huge market demands and create a continued interest in the production of costeffective, more specific, less immunogenic and stable formulations which can cater both the clinical and food processing requirements. The current review article approaches the process parameters of submerged and solid-state fermentation strategies for the microbial production of the L-asparaginase from diverse sources, genetic engineering approaches used for the production of L-asparaginase enzyme and major applications in clinical and food sectors. The review also addresses the immunological issues associated with the L-asparaginase usage and the immobilization strategies, drug delivery systems employed to circumvent the toxicity complications are also discussed. The future prospects for microbial Lasparaginase production are discussed at the end of the review article.

The goal of the review is description of the main characteristics of creatinine deiminase (CDI), an important bioanalytical tool for creatinine (Crn) assay. Crn is an essential metabolite for diagnostics of kidney disfunction and some other diseases, a biomarker to control the hemodialysis procedure, as well as an important analyte for sport medicine (estimation of general physiological status of athletes). We have described the important sources for CDI isolation, cloning of the corresponding gene, the construction of microbial recombinant strains, overproducing CDI, and characteristics of the enzyme from different microorganisms. There are reviewing also the new bioanalytical methods for quantitative determination of Crn, including enzymatic ones based on using CDI.

Pseudomonas aeruginosa is a non-fermentative, gram-negative bacterium that is one of the most common pathogens responsible for hospital-acquired infections worldwide. The management of the infections caused by P. aeruginosa represents a huge challenge in the healthcare settings due to the increased emergence of resistant isolates, some of them resistant to all the currently available antimicrobials, which results in elevated morbimortality rates. Consequently, the development of new therapeutic strategies against multidrug-resistant P. aeruginosa is urgent and needful. P. aeruginosa is wellrecognized for its extreme genetic versatility and its ability to produce a lush variety of virulence factors. In this context, pseudolysin (or elastase B) outstands as a pivotal virulence attribute during the infectious process, playing multifunctional roles in different aspects of the pathogen-host interaction. This protein is a 33-kDa neutral zinc-dependent metallopeptidase that is the most abundant peptidase found in pseudomonal secretions, which contributes to the invasiveness of P. aeruginosa due to its ability to cleave several extracellular matrix proteins and to disrupt the basolateral intercellular junctions present in the host tissues. Moreover, pseudolysin makes P. aeruginosa able to overcome host defenses by the hydrolysis of many immunologically relevant molecules, including antibodies and complement components. The attenuation of this striking peptidase therefore emerges as an alternative and promising antivirulence strategy to combat antibiotic-refractory infections caused by P. aeruginosa. The anti-virulence approach aims to disarm the P. aeruginosa infective arsenal by inhibiting the expression/activity of bacterial virulence factors in order to reduce the invasiveness of P. aeruginosa, avoiding the emergence of resistance since the proliferation is not affected. This review summarizes the most relevant features of pseudolysin and highlights this enzyme as a promising target for the development of new anti-virulence compounds.