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Nanostructured Cellular Biomolecules and Their Transformation in Context of Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
Amino acids are linked together in a polypeptide chain by peptide bond. The linkage between amino acids occurs through simple condensation reaction between the α-amino groups of one amino acid with α-carboxyl group of another amino acid with the release of a water molecule. Linked amino acids in a polypeptide chain are called amino acid residues. The free amino group and carboxyl group at the opposite ends of peptide chain are called the N-terminal and C-terminal, respectively. During protein synthesis, polypeptide chain formation starts from N-terminal of amino acid (usually methionine) and continues toward the C-terminals by adding one amino acid at a time. Depending on the number of amino acids linked together, they are termed as dipeptide, tripeptide, oligopeptide, and polypeptide. Dipeptide contains two amino acids linked by one peptide bond. Therefore, each peptide chain has one free amino and carboxyl group at opposite ends (Moran et al. 2012).
Protein structure prediction
Published in A. K. Haghi, Lionello Pogliani, Devrim Balköse, Omari V. Mukbaniani, Andrew G. Mercader, Applied Chemistry and Chemical Engineering, 2017
It is the linear sequence of amino acids, which is chemically a polypeptide chain, joined by peptide bonds.1, 2 Peptide bonds are covalent in nature and many peptide bonds form polypeptide chain. These peptide bonds are formed during translation (protein synthesis). There are two terminals in any polypeptide chain C-terminus (carboxyl group) and the N-terminus (amino group). Counting of residues always starts at the N-terminal end (NH2 group), which is the end where the amino group is not involved in a peptide bond. The primary structure of a protein is determined by the gene corresponding to the protein. Sequence of nucleotide, that is, DNA forms mRNA which is known as transcription and when mRNAs are synthesizing proteins, it is known as translation and the whole process is known as central dogma. Each protein has its specific amino acid sequence.3, 4 These specific orders of amino acids are unique for every protein and they determine the structure and function of the proteins. Order of amino acids can be determined by Edman degradation method r mass spectrometry. There are approximately 10,000 different proteins in any human being and all are composed from just 20 different types of amino acids residues.5 Protein formation is followed by post-translational modifications which include disulphide bond formation, addition of glycosyl molecules (glycosylation), and phosphorylation.
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Typically, protein expression vectors include tags for purification and immunohistological identification of the heterologously expressed protein. In most cases, tags are appended to the N-terminus or C-terminus of the protein during subcloning into the protein expression vector. If C-terminal tags are to be appended to the target protein, then the protein must be inserted into the vector without a stop codon. When the vector contains N-terminal tags, care must be taken to insert the gene in frame with the start codon before the N-terminal tags. If a vector contains C-terminal tags that one does not want to append to the protein of interest, it is typically sufficient to leave the native stop codon in place during the subcloning. However, if the vector contains N-terminal tags that one does not want to append to the protein of interest, these tags must be removed from the vector during subcloning. While tags are typically inserted at only either the N-terminus or the C-terminus of the protein, it is possible to insert tags for purification or immunohistological identification into the core of the protein. This strategy has been successfully applied to purification tags inserted into the extracellular loops of multipass transmembrane proteins.
Molecular packing handedness dominated by the chirality of the lactic acid residue near the liquid crystalline core
Published in Liquid Crystals, 2021
Xiaoqing Wu, Baining Ni, Limin Wu, Yongmin Guo, Yi Li, Baozong Li, Yonggang Yang
Recently, the self-assembly behaviours of liquid crystals in solvents arouse much attention [20–23]. The molecules can organise into varieties of nanostructures through oleophobic/hydrophobic associations, hydrogen bondings and π-π interactions. Although a lot of liquid crystals with single chiral centres have been reported, the reports on dichiral liquid crystals are still limited [24–32]. It was found that the dichiral compounds with two linked lactate groups exhibit many kinds of chiral mesophases. Moreover, the mesophase structures were dominated by the chirality [32]. During the last decade, varieties of chiral low-molecular-weight gelators have been reported. Generally, they can self-assemble into twisted nanoribbon, coiled nanoribbon, nanotube and helical bundle [23–35]. With increasing the ageing time, a structural evolution from twisted nanoribbon to coiled nanoribbon, and then to nanotube was identified [34]. Based on the self-assembly behaviours of dichiral low-molecular-weight gelators, several series of homogeneous lipodipeptides have been systematically studied by our group [36,37]. A ‘C-terminal determination’ rule was found. The handedness of the organic self-assemblies was controlled by the chirality of the amino acid at the C-terminal. Herein, two series of liquid crystals with two linked lactate groups were synthesised, which can self-assemble into nanotubes or twisted nanoribbons in methanol. Both the molecular chirality and the molecular packing handedness in organic self-assemblies are dominated by the chirality of the lactic acid residue near the liquid crystalline core.
Rapid label-free detection of E. coli using a novel SPR biosensor containing a fragment of tail protein from phage lambda
Published in Preparative Biochemistry and Biotechnology, 2018
In an effort to overcome this issue and also to develop an SPR biosensor, a His-tag was used here to produce a fusion protein of C-terminal fragment of J. This strategy was based on the assumption that a small ligand might have less steric hindrance effects and increase the SPR response to bacterial attachment, as described in a previous report.[15] The C-terminal portion of J protein was cloned and a (His)6-tag at its N-terminus (6HN-J) was constructed. The purified 6HN-J protein was examined for its binding specificity to E. coli by various approaches, such as ELISA, dot blot, TEM, and an in vivo adsorption assay. Following its validation, the fusion protein was exploited in an SPR biosensor.
Cloning, expression and characterization of a HER2-alpha luffin fusion protein in Escherichia coli
Published in Preparative Biochemistry and Biotechnology, 2019
Farzaneh Barkhordari, Nooshin Sohrabi, Fatemeh Davami, Fereidoun Mahboudi, Yeganeh Talebkhan Garoosi
The amino acid sequence of alpha luffin protein was extracted from UniProtKB-Q00465. Anti-HER2 scFv was originated from the variable regions of the light and heavy chains of anti-HER2 monoclonal antibody, Trastuzumab, obtained from the drug bank database (accession number: DB00072) and published patent (US20060275305A1). The amino acids were converted into the corresponding nucleotide sequences and were linked together using a flexible fragment encoding 15 amino acid peptide linker (Gly4Ser)3. For confirmation and easy purification of the recombinant fusion protein, a Histidine tag was designed at N-terminal end of the construct. Due to the dominant expression of Cathepsin B, lysosomal cysteine protease in breast cancer cell lines compared to the normal cells (Human Protein Atlas), its sensitive cleavage site (GFLG) was inserted immediately after the peptide linker sequence for efficient cleavage of the toxic part from anti-HER2 scFv molecule upon its cellular internalization. A conserved hydrophobic KDEL recognition motif was also incorporated at C-terminal end of the designed construct to increase the endosomal scape of the toxic domain to the trans-Golgi network as an endoplasmic reticulum retention signal. Nucleotide fragments described above were assembled together (1596 bp) and converted into the amino acid sequence (532 amino acids). The open reading frame (ORF) of the whole amino acid sequence encoding the fusion protein was checked using Gene Runner (V. 6.0). The codon optimized encoding cDNA fragment was synthesized and subcloned into the expression vector, pET28a (Novagen, USA) at NcoI and HindIII restriction sites under the control of T7 promoter and transformed into E. coli BL21 (DE3), BL21 (DE3) pLysS, Rosetta (DE3) pLysS, and HI-Control BL21 (DE3) (Merck) host cells.