Amino acid sequences synthesis

Parker, F., Migliore-Samour, D., Floc h, F., Zerial, A., Werner, G.H., Jolles, J., Casaretto, M., Zahn, H., and Jolles, P. 1984. Immunostimulating hexapeptide from human casein, amino acid sequence, synthesis and biological properties. Eur. J. Biochem. 145, 677-682. 

As described in the preceding sections protein synthesis involves transcription of the DNA to rtiRNA followed by translation of the mRNA as an amino acid sequence In addition to outlining the mechanics of transcription we have described the relationship among mRNA codons tRNA anticodons and ammo acids... 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

Biosynthesis. CRE is derived from a precursor of 196 amino acids (84,85). This gene contains one copy of CRE, which is flanked by double basic amino acids. The amino acid sequence of the CRE precursor suggests that it may arise from proteins related to POMC and neurophysins (31). The CRE precursor contains a cAMP responsive element which aHows stimulation of mRNA synthesis when intraceHular levels of cAMP are increased (86). 

The synthesis of virtually all proteins in a cell begins on ribosomes in the cytosol (except a few mitochondrial, and in the case of plants, a few chloroplast proteins that are synthesized on ribosomes inside these organelles). The fate of a protein molecule depends on its amino acid sequence, which can contain sorting signals that direct it to its corresponding organelle. Whereas proteins of mitochondria, peroxisomes, chloroplasts and of the interior of the nucleus are delivered directly from the cytosol, all other organelles receive their set of proteins indirectly via the ER. These proteins enter the so-called secretory pathway (Fig. 1). 

The cell must possess the machinery necessary to translate information accurately and efficiently from the nucleotide sequence of an mRNA into the sequence of amino acids of the corresponding specific protein. Clarification of our understanding of this process, which is termed translation, awaited deciphering of the genetic code. It was realized early that mRNA molecules themselves have no affinity for amino acids and, therefore, that the translation of the information in the mRNA nucleotide sequence into the amino acid sequence of a protein requires an intermediate adapter molecule. This adapter molecule must recognize a specific nucleotide sequence on the one hand as well as a specific amino acid on the other. With such an adapter molecule, the cell can direct a specific amino acid into the proper sequential position of a protein during its synthesis as dictated by the nucleotide sequence of the specific mRNA. In fact, the functional groups of the amino acids do not themselves actually come into contact with the mRNA template. 

In the synthesis of polypeptides with biological activity on a crosslinked polymer support as pioneered by Merrifield (1 2) a strict control of the amino acid sequence requires that each of the consecutive reactions should go virtually to completion. Thus, for the preparation of a polypeptide with 60 amino acid residues, even an average conversion of 99% would contaminate the product with an unacceptable amount of "defect chains". Yet, it has been observed (13) that with a large excess of an amino acid reagent —Tn the solution reacting with a polymer-bound polypeptide, the reaction kinetics deviate significantly from the expected exponential approach to quantitative conversion, indicating that the reactive sites on the polymer are not equally reactive. 

In addition to the enzyme s amino acid sequence, other parameters can affect the outcome of a biocatalytic process. For instance, a similar outcome in the aforementioned DERA-catalyzed statin synthesis was achieved by process improvements [21]. Using a thermostable variant of DERA (thermostability generally correlates well with tolerance to high concentrations of organic reagents or cosolvents), and fed-batch conditions, an efficient process that overcame sensitivity to high concentrations of chloroacetaldehyde was developed. 

Genetic code Sequence of nucleotides along the DNA and coded in triplets (codons) along the mRNA that determines the sequence of amino acids in protein synthesis. The DNA sequence of a gene can be used to predict the mRNA sequence, and subsequently to predict the amino acid sequence. 

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