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Basic Molecular Cloning of DNA and RNA
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Many hundreds of plasmids are available commercially; the primary suppliers are listed at the end of this chapter. Some plasmids are empty except for a resistance gene and a promoter; these are known as cloning vectors (Figure 2.1a). More complex plasmids are intended for expression of the protein in E. coli, in which case they are called bacterial expression vectors (Figure 2.1b). Expression vectors for eukaryotic cells (yeast, mammalian cells, plants, etc.) contain an entire expression sequence that permits the gene to express in these cells (Figure 2.1c). Plasmids for expression of genes in bacteria other than E. coli and its relatives also usually use such an expression sequence, and E. coli is used as an intermediate because of its ease of use for cloning; this will be discussed further in Chapter 3.
Recombinant DNA technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
All vectors used for propagation of DNA inserts in a suitable host are called cloning vectors. However, when a vector is designed for the expression or production of the protein specified by the DNA insert, it is called an expression vector. As a rule, such vectors contain at least the regulatory sequences such as promoters, operators, and ribosome-binding sites having an optimum function in the chosen host. When a eukaryotic gene is to be expressed in a prokaryote, the eukaryotic coding sequence has to be placed over prokaryotic promoter and the ribosome-building site since the regulatory sequences of eukaryotes are not recognized in prokaryotes. In addition, eukaryotic genes, as a rule, contain introns (noncoding regions) present within their coding regions. These introns must be removed from the DNA insert to enable the proper expression of eukaryotic genes, since prokaryotes lack the machinery needed for their removal from the RNA transcripts. When eukaryotic genes are isolated as cDNA, they are intron free and, therefore, suitable for expression in prokaryotes. Expression vectors can be constructed by allowing the synthesis of fusion proteins—which comprise amino acids encoded by a sequence—in the vector and those encoded by the DNA insert (translational fusion). Another way to construct expression vectors is by permitting the synthesis of pure proteins encoded exclusively by the DNA inserts (transcriptional fusion). Some examples of the first strategy, which produces fusion proteins, are the expression of rat insulin, a rat growth hormone, structural protein VP1 of foot and mouth disease virus, and human growth hormone. On the other hand, some examples of the second strategy, which produces unique proteins, are the rabbit (3-globin, small-T antigen of SV40, human fibroblast interferon, and human IGF-I protein. It may be pointed out that in the case of translational fusion, the undesired amino acids encoded by the vector sequence must be removed from the fusion proteins by a suitable chemical cleavage. Several other problems arise when eukaryotic genes are expressed in a prokaryotic system such as removal of signal sequences from precursor proteins to obtain active mature protein molecules. Various strategies are being rapidly devised to effectively overcome these problems.
Recombinant DNA Technology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
All vectors used for propagation of DNA inserts in a suitable host are called cloning vectors. However, when a vector is designed for the expression or production of the protein specified by the DNA insert, it is called an expression vector. As a rule, such vectors contain at least the regulatory sequences such as promoters, operators, and ribosomal binding sites having an optimum function in the chosen host. When a eukaryotic gene is to be expressed in a prokaryote, the eukaryotic coding sequence should be placed over the prokaryotic promoter and the ribosome building site since the regulatory sequences of eukaryotes are not recognized in prokaryotes. In addition, eukaryotic genes, as a rule, contain introns (noncoding regions) present within their coding regions. These introns must be removed from the DNA insert to enable the proper expression of eukaryotic genes, since prokaryotes lack the machinery needed for their removal from the RNA transcripts. When eukaryotic genes are isolated as cDNA, they are intron-free and therefore suitable for expression in prokaryotes. Expression vectors can be constructed by allowing the synthesis of fusion proteins—which are composed of amino acids encoded by a sequence—in the vector and those encoded by the DNA insert (translational fusion). Another way to construct expression vectors is by permitting the synthesis of pure proteins encoded exclusively by the DNA inserts (transcriptional fusion). Some examples of the first strategy, which produces fusion proteins, are the expression of rat insulin, a rat growth hormone, structural protein VP1 of foot and mouth disease virus, and human growth hormone. On the other hand, some examples of the second strategy, which produces unique proteins, are the rabbit β-globin, small t-antigen of SV40, human fibroblast interferon, and human IGF-I protein. It may be pointed out that in the case of translational fusion the undesired amino acids encoded by the vector sequence must be removed from the fusion proteins by a suitable chemical cleavage. Several other problems arise when eukaryotic genes are expressed in a prokaryotic system, such as removal of signal sequences from precursor proteins to obtain active mature protein molecules. Various strategies are being rapidly devised to effectively overcome these problems.
Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells
Published in Preparative Biochemistry & Biotechnology, 2018
Hadi Bayat, Saghar Hoseinzadeh, Eśhagh Pourmaleki, Roshanak Ahani, Azam Rahimpour
Expression vector engineering strategies have been shown to be highly effective for improving monoclonal antibody expression level and its stability in mammalian cells. In addition, it has been shown that expression cassette design can affect the ratio of LC:HC polypeptides.[7] The aim of the current study was to compare different vector design strategies for the expression of IgG1 mAb in CHO cells. Here the antibody productivity was evaluated in both transient and stable expressions using three common vector systems including two independent vectors for the expression of LC and HC, bicistronic single-vector and dual-promoter single-vector systems. In addition, the antibody expression level was analyzed in the clonal cells derived from each cell pool.
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
GPC3-N terminal protein (1005 bp) was successfully polymerized from GPC3 template DNA (Yiqiao Shenzhou, Beijing) using preoptimized primers (Sangon Biotech) in thermocycler reaction. The PCR products were loaded on 0.8–1% agarose gel and the bands showed correct orientation of targeted GPC3 N-terminal protein as shown in Figure 1a. PCR amplification of the GPC3 N-terminal protein gene before optimization was optimized and bands of 1005 bp were recorded. The products were recovered by purification kit and ligated to pMD18-T cloning vector to construct the recombinant pMD18T-GPC3-N terminal vector as shown in Figure 1b. The recombinant vector was transformed into competent E. coli DH5α and allowed to grow on Amp-positive plates. The selected positive colonies were inoculated in a 5-ml LB tubes (Supplemented with 100 µg/ml ampicillin). The extracted plasmid was digested using restriction enzymes, to identify the targeted sequence of 1005 bp GPC3 N-terminal sequences as shown in Figure 1c. The second polymerization reaction was repeated using 1005 bp, deprived six proline amino acids, using different annealing temperatures (50, 55, and 65°C). Optimum products of 987 bp were obtained at 65°C as shown in Figure 1d,e. The amplified products were transformed into E. coli DH5α and plated on ampicillin-positive plates. A single colony was inoculated in LB tubes and incubated at 37°C followed by plasmid extraction. The successful recombinant formation was verified by PCR and enzyme digestion. Targeted sequence-deprived proline residues were observed by sequence determination (Sangon Biotech) and found correct recombinant construct. Both these recombinant plasmids were further processed for development of expression vector.
Development of an improved lentiviral based vector system for the stable expression of monoclonal antibody in CHO cells
Published in Preparative Biochemistry and Biotechnology, 2019
Omid Mohammadian, Masoumeh Rajabibazl, Es’hagh Pourmaleki, Hadi Bayat, Roshanak Ahani, Azam Rahimpour
The choice of expression vector and its regulatory elements can significantly affect transgene expression in mammalian cells. Lentiviralbased vectors provide attractive tools for in vivo and in vitro gene delivery to mammalian cells due to their high transduction efficiency, and stable-long term expression of transgene.[31]