Alanine gene cloning

Using directed mutation of the cloned receptor gene, Lys 1018 in the ATP-binding part of the tyrosine kinase domain was replaced by alanine. This caused a loss both of kinase activity and of biologic response to insulin.362 Thus, both the tyrosine kinase activity and autophosphorylation appear essential. If so, aggregation of two or more receptors may increase the extent of autophosphorylation and initiate a response. 

E Daub, LE Zawadzke, D Botstein, CT Walsh. Isolation, cloning, and sequencing of the Salmonella typhimurium ddlA gene with purification and characterization of its product, D-alanine D-alanine ligase (ADP forming). Biochemistry 27 3701-3708, 1988. 

Alanine racemase was purified and cloned form various sources. Interestingly, two distinct genes were identified in a number of genome sequences, for example, E. coli, BadUus suhtilis. Pseudomonas aeru nosa, and so on. 

Hoppensack, A., Rehm, B.H.A., and Steinbiichel, A. (1999) Analysis of 4-phosphopantetheinylation of polyhydroxybutyrate synthase from Ralstonia eutropha generation of beta-alanine auxotrophic Tn5 mutants and cloning of the panD gene region. J. Bacteriol., 181, 1429-1435. 

Human SOD was also expressed in the periplasmic space of E. coli using a secretion vector that allows the protein to be accumulated in the periplasmic space. In 1988 the HSOD gene was cloned in a secretion vector pIN-III OmpA (142) On induction of gene expression the protein was produced at a level of 10% of total cellular protein and was recovered through osmotic shock as the major component of the periplasmic fraction. Although the N-terminal alanine is not acetylated, it is practically indistinguishable from the acetylated form (see Table Til). 

Heaton MP, Neuhaus FC. Biosynthesis of D-alanyl-lipoteichoic acid Cloning, nucleotide sequence, and expression of the LoctobadDus casei gene for the D-alanine-activating enzyme. J Bacteriol 1992 174 4707-4717. 

Figure 3 Sequences of selected conjugates, (a) Designed sequence of peptides attached to fusion proteins as expressed by phage library 1. Processing of the leader sequence upon secretion of the protein is expected to give a peptide with an N-terminal alanine, two random six-amino-acid sequences flanked by three cysteines and a Gly-Gly-Ser-Gly linker that connects the peptide to the gene-3-protein. (b,c) Amino acid sequences of conjugates selected with human plasma kallikrein (b) and cathepsin G (c) with corresponding inhibitory activity. Only those clones with sequence similarities or that were isolated multiple times are displayed. Residues of similar character are highlighted in color. ND, not determined.

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