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Cloning of genes for protein expression
Published in Raimund J. Ober, E. Sally Ward, Jerry Chao, Quantitative Bioimaging, 2020
Raimund J. Ober, E. Sally Ward, Jerry Chao
Sometimes the fluorescent protein gene needs to be attached at the N-terminus of the protein. This is slightly more complicated since if the protein is to be secreted or inserted into a membrane, the fluorescent protein gene needs to be inserted between the end of the leader peptide that directs the protein to the correct destination in the cell and the beginning of the mature end of the protein (the leader peptide is usually cleaved from the mature protein once it has reached the appropriate location). This step can be achieved using one of several different molecular biology approaches. One approach is to use the PCR to append restriction sites between the ends of the leader peptide and mature protein and to then append the same restriction sites (using the PCR) to the ends of the fluorescent protein gene.
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 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.
Solid-phase synthesis of sulfur containing heterocycles
Published in Journal of Sulfur Chemistry, 2018
The thiomethyl thiazolyl macrocyclic peptidomimetics were synthesized from resin-bound orthogonally protected Fmoc-Cys-(Trt)-OH via Fmoc de-protection and standard repetitive Fmoc-amino acid couplings yielding the linear tripeptide (Scheme 24). The formed N-terminal free amine was reacted with Fmoc-isothiocyanate. After the Fmoc de-protection, the thioureas were reacted with 1,3-dichloroacetone. Then Hantzsch's cyclocondensation provided the resin-bound chloro methyl thiazolyl peptide. The 5% trifluoroacetic acid in CH2Cl2 was utilized for the de-protection of Trt group. The resin underwent an SN2 intramolecular cyclization in the presence of a solution of cesium carbonate in dimethylformamide. The resin was cleaved with HF/anisole to afford desired thiazolyl thioether cyclic peptides in high purity and good yield [19,55].
Identification and characterization of candidates involved in production of OMEGAs in microalgae: a gene mining and phylogenomic approach
Published in Preparative Biochemistry and Biotechnology, 2018
Vikas U. Kapase, Asha A. Nesamma, Pannaga P. Jutur
To determine the putative subcellular localization of these candidate proteins, three different target prediction programs namely TargetP,[19] ngLOC,[20,21] and Cello[22] were employed based on their terminology and predictions. TargetP predicts the localization by examining presence of the N-terminal presequences, chloroplast transit peptides (cTP), mitochondria targeting peptides (mTP), and secretary pathway signal peptide (SP),[19] whereas the ngLOC program is based on n-gram-based Bayesian approach, which predicts subcellular protein localization in ten different subcellular organelles.[21] The Cello program identifies the n-peptide composition based on a two-level support vector machine (SVM) classifier and the homology search method for the sequence annotation of subcellular localization prediction.[22]
Polyclonal antibody production against rGPC3 and their application in diagnosis of hepatocellular carcinoma
Published in Preparative Biochemistry and Biotechnology, 2018
Shenghao Wang, Muhammad Kalim, Keying Liang, Jinbiao Zhan
Escherichia coli expression system possesses a potent source of recombinant protein production. But in the expression of exogenous protein, especially in the expression of eukaryotic proteins, there are still many problems that limit expression level, no expression or lost biological activity due to structural change and protein modification translation. We have achieved the high expression level of GPC3 N-terminal protein by deleting the 18 bases of six proline residues from GPC3 N-terminal protein. Successive washing methodology and refolding techniques provide an active production of recombinant proteins. Thereafter, the rGPC3 N-terminal protein was used as an antigen to immunize New Zealand male rabbits to obtain anti-GPC3 polyclonal antibody. The polyclonal antibody has shown specific binding interactions to the GPC3 surface antigen on primary liver cancer cell lines (Huh7 and HepG2). Immunohistochemistry assay also proves that polyclonal antibody can combine with GPC3 in HCC cancer tissue. In addition, the recombinant GPC3 N-terminal protein provides a basis for the screening of monoclonal antibodies for HCC-targeted therapy. The findings here will be useful for the production of valuable antibodies which can be used for cancer diagnosis and immunotherapy.