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Proteins and proteomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
The primary structure is held together by covalent or peptide bonds, which are made during the process of protein biosynthesis or translation. These peptide bonds provide rigidity to the protein. The two ends of the amino acid chain are referred to as the C-terminal end or carboxyl terminus (C-terminus) and the N-terminal end or amino terminus (N-terminus) based on the nature of the free group on each extremity. The various types of secondary structures are defined by their patterns of hydrogen bonds between the main-chain peptide groups. However, these hydrogen bonds are generally not stable by themselves, since the water—amide hydrogen bond is generally more favorable than the amide—amide hydrogen bond. Thus, the secondary structure is stable only when the local concentration of water is sufficiently low, for example, in the molten globule or fully folded states. Similarly, the formation of molten globules and tertiary structure is driven mainly by structurally nonspecific interactions such as the rough propensities of the amino acids and hydrophobic interactions. However, the tertiary structure is fixed only when the parts of a protein domain are locked into place by structurally specific interactions, which include ionic interactions (salt bridges), hydrogen bonds, and the tight packing of side chains. The tertiary structure of extracellular proteins can also be stabilized by disulfide bonds, which reduce the entropy of the unfolded state. Disulfide bonds are extremely rare in cytosolic proteins since the cytosol is generally a reducing environment.
Naturally Occurring Polymers—Animals
Published in Charles E. Carraher, Carraher's Polymer Chemistry, 2017
Proteins are synthesized in nature from the N-terminus to the C-terminus. While all the amino acids have only one variation, with that variation being a lone substituent on the alpha-carbon, this variation is sufficient to produce the variety of proteins critical to life. Some of these contain an “excess” of acid groups, such as aspartic acid, while others contain one or more additional basic groups such as arginine. Still others possess sulfur-containing moieties, namely, cysteine and methionine, that account for the need for sulfur to be present in our diets and eventually end up in our fossil fuels, producing sulfur oxides on burning. All possess nitrogen-containing moieties and these also eventually end up in our fossil fuels and produce nitrogen oxides on burning. The presence of these two elements is then connected to acid rain production.
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.
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.