Monomers genes/sequence

Transformation of E. coli, Production of Much Monomer Gene and Verification of Monomer Gene Sequence... 

Table 1 Microbial Biosynthesis Basic Monomer Gene Sequences... 

Selenoprotein P is a glycoprotein the sequence of the protein moiety within it, having a molecular mass of about 41 kDa, was deduced from the nucleotide sequence of the rat gene. It is unique since, dependent on the organism, it contains more than a dozen selenocysteine residues per monomer. The sequence also contains histidine- and cysteine-rich stretches, which could be involved in metal binding. Other... 

While biological synthesis of polymers confers many advantages, it also imposes some limitations. As described previously, there are several issues that must be considered when designing the monomer and concatamer gene sequences. Furthermore, the potential toxicity of the genetically engineered polymer to the expression host, and the resultant effect on the integrity of the product must be considered. 

Hius one designs a DNA sequence for a basic monomer gene, including the sequences of the restriction sites as required for the protein to be expressed and the plasmid to be used, as included in Figure 9.2. The next step is to... 

Figure 9.2. Diagram showing base sequences for form the multimer gene for expression of the elastic restriction sites at ends of a basic monomer gene protein-based polymer [(GVGVP)io] (GVGVP). encoding for (GVGVP)io and for concatenation to (Reproduced with permission from Urry et al. )...

An appropriate plasmid and strain of E. coli are chosen, for example, for the transformation and growing up of the transformed E. coli containing many plasmids, each plasmid with one copy each of the monomer gene. The plasmids are harvested, the monomer genes excised by the appropriate restriction enzyme(s), and the sequence of the monomer gene verified. 

Having verified that the repeating sequence of the expressed protein agrees with the sequenced monomer gene, it is now to be determined how many repeating units constitute the expressed protein-based polymer, that is, the... 

In humans, the structural gene locus is on chromosome 19 (M17), and the gene spans over 40 kilobases (kb) including 18 exons and 17 introns (W2, X2). Neu-roleukin, a protein that acts as both a neurotrophic factor and a lymphokine, has been isolated from mouse salivary glands (G7), and subsequently the primary structure of neuroleukin was found to be identical to that of GPI by comparison of the cDNA sequences (C7, FI). The cDNA sequence encodes 558 amino acid residues. The enzyme consists of two identical subunits with a molecular weight of approximately 63,000 and neuroleukin is active as a monomer. 

A synthetic DNA cassette was designed that encoded the repeat sequence Lys-25 (Figure 2). This DNA monomer was synthesized, sequenced, and self-ligated to afford a mixture of DNA concatamers. A 3,000 base pair concatameric gene was iolated from this mixture, which encoded a polymer of Lys-25 with a molecular mass of approximately 90 kD. Bacterial expression of the concatameric gene afforded a protein of the expected molecular mass that accumulates to high levels in E. coli (Figure 3). 

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