Rohdehuang9687

Expressed protein ligation allows for the attachment of a chemically labeled peptide to the N- or C-terminus of a recombinant protein. In this book chapter, the practical considerations involved in using this protein engineering technology are described. In particular, approaches used to design optimal ligation sites are discussed. In addition, several methods used to generate the reactive fragments required for EPL are highlighted in practical details. Finally, strategies that one can implement to achieve efficient ligation reactions are presented.The autocatalytic process of protein splicing is facilitated by an intein, which interrupts flanking polypeptides called exteins. The mechanism of protein splicing has been studied by overexpression in E. coli of intein fusion proteins with nonnative exteins. Inteins can be used to generate reactive α-thioesters, as well as proteins with N-terminal Cys residues, to facilitate expressed protein ligation. As such, a more detailed understanding of the function of inteins can have significant impact for biotechnology applications. Here, we provide biochemical methods to study splicing activity and NMR methods to study intein structure and the catalytic mechanism.In recent years, split inteins have seen widespread use as molecular platforms for the design of a variety of peptide and protein chemistry technologies, most notably protein ligation. The development of these approaches is dependent on the identification and/or design of split inteins with robust activity, stability, and solubility. Here, we describe two approaches to characterize and compare the activities of newly identified or engineered split inteins. The first assay employs an E. coli-based selection system to rapidly screen the activities of many inteins and can be repurposed for directed evolution. The second assay utilizes reverse-phase high-performance liquid chromatography (RP-HPLC) to provide insights into individual chemical steps in the protein splicing reaction, information that can guide further engineering efforts. These techniques provide useful alternatives to common assays that utilize SDS-PAGE to analyze splicing reaction progress.Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size. This methodology has been developed based on the knowledge obtained from protein splicing. Protein splicing is a multistep biochemical reaction that includes the concomitant cleavage and formation of peptide bonds carried out by self-processing domains named inteins. The natural substrates of protein splicing are essential proteins found in intein-containing organisms; inteins are also functional in nonnative frameworks and can be used to alter nearly any protein's primary amino acid sequence. Accordingly, different reactivity features of inteins have been largely exploited to manipulate proteins in countless methods encompassing fields from biochemical research to the development of biotechnological applications including the study of disease progression and validation of potential drug candidates. Here, we review almost three decades of research to uncover the chemical and biochemical enigmas of protein splicing and the development of inteins as potent protein engineering tools.Expressed protein ligation is a method of protein semisynthesis and typically involves the reaction of recombinant protein C-terminal thioesters with N-cysteine containing synthetic peptides in a chemoselective ligation. The recombinant protein C-terminal thioesters are produced by exploiting the action of nature's inteins which are protein modules that catalyze protein splicing. This chapter discusses the basic principles of expressed protein ligation and recent advances and applications in this protein semisynthesis field. Comparative strengths and weaknesses of the method and future challenges are highlighted.The aim of this study was to evaluate the impact of increased shadow supply in integrated crop-livestock-forest systems on in vitro embryonic development and physiological parameters related to stress response in Nellore heifers (Bos indicus). For the study, animals (n = 16) were randomly divided into two groups and kept in areas with different afforestation systems, the integrated crop-livestock-forest (ICLF) and the integrated crop-livestock (ICL) system. The microclimate of the ICLF system provided better comfort conditions than ICL. No differences of respiratory rate, rectal temperature, cortisol, T3, T4, oocyte quality, and cleavage rate between the systems were verified. A higher blastocyst rate was observed in the ICLF (p  less then  0.05). The results demonstrate that Nellore heifers managed in ICLF during summer in Midwest of Brazil showed higher production of in vitro embryos, without typical changes in its physiological parameters. The results observed in the present study indicate that zebu females are able to respond satisfactorily to the intense heat conditions; however, we believe that the long period to which these animals are exposed to these conditions interferes in the oocyte competence and embryo development.The aim of the present study was to estimate the (co)variance components and genetic parameters for various growth traits (weight at birth (BWT) and 3 (WT3), 6 (WT6), 9 (WT9), and 12 (WT12) months of age), average daily gain (ADG1, 0-3; ADG2, 3-6; and ADG3, 6-12 months of age), and Kleiber's ratio (KR1ADG1/WT30.75 and KR2ADG2/WT60.75) by using records of 526 lambs of 41 sires and 186 dams in Harnali Sheep maintained at Department of Animal Genetics and Breeding, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar (Haryana), India for the period of year 2014-2019. HSP (HSP90) inhibitor Restricted maximum likelihood procedure (REML) was employed for estimation of covariance components and genetic parameters by considering direct effects with or without maternal effects. The estimates of direct heritability for BWT, WT3, WT6, WT9, WT12, ADG1, ADG2, ADG3, KR1, and KR2 were 0.10, 0.45, 0.32, 0.36, 0.23, 0.43, 0.02, 0.001, 0.38, and 0.02, respectively. It was observed that maternal effects had significant influence on BWT trait only, and corresponding estimate of maternal heritability was 0.