Lysin sequences

Humalog (Insulin lispro) Recombinant short-acting human insulin analogue produced in E. coli Engineered inversion of native B28-B29 proline-lysine sequence Eli Lilly 1996 (USA and EU)... 

Insulin Lispro was the first recombinant fast-acting insulin analogue to gain marketing approval (Table 8.3). It displays an amino acid sequence identical to native human insulin, with one alteration — an inversion of the natural proline lysine sequence found at positions 28 and 29 of the insulin jS-chain. This simple alteration significantly decreased the propensity of individual insulin molecules to self-associate when stored at therapeutic dose concentrations. The dimerization constant for Insulin Lispro is 300 times lower than that exhibited by unmodified human insulin. Structurally, this appears to occur as the change in sequence disrupts the formation of inter-chain hydrophobic interactions critical to self-association. 

This first study encouraged us to continue to obtain additional lysin sequences. We found that ethanol fixation (50%-90%) of fragments of abalone testis preserved the lysin mRNA, making possible procurement of testis samples from approximately 30 species world wide. Following the synthesis of universal lysin primers in the 3 and 5 untranslated regions of lysin cDNA, it was a simple process to obtain lysin sequences from an additional 25 species using RT PCR (Lee et al., 1995 Lee and Vacquier, 1995). 

Primers were made to obtain the full length sequences of five species by PCR (Swanson and Vacquier, 1995b). Instead of presenting an alignment of all 27 lysin sequences and the five 18K sequences, we will present the sequences of both proteins from five species of California abalone (Figure 9 Vacquier et al., 1997). The sequences of both acrosomal proteins are known for four species. Haliotis kamtschatkana (known for lysin) and H. assimilis (known for 18K) are considered to be comparable, closely related species. 

When the lysin sequences of the first seven species were obtained, we were impressed at how much divergence had occurred between their primary structures (Figure 9 Table 1). We also discovered that amino acid replacement was mainly nonconservative regarding the class of residue replaced. Next, we made pairwise comparisons of the aligned cDNA sequences and scored the numbers of amino acid altering (nonsynonymous) and silent (synonymous) nucleotide changes in the 21 pairwise comparisons of the seven sequences. The data (Table 2) showed that the vast majority of codon differences between any two lysins are amino acid altering. For example, in the comparison of mature red and pinto abalone lysins of 136 codons, 25 of the codon differences are nonsynonymous and only one is silent (Lee and Vacquier, 1992). 

InsuHn Hspro (sold under the trade names Humalog and Liprolog) exemplifies engineered short-acting insuHn products. This product displays an amino acid sequence identical to native human insuHn, with the exception that the natural pro-Hne-lysine sequence characteristics of positions 28 and 29 of the insuHn B chain have been reversed. The sequence inversion leads to local conformational changes, eHminating hydrophobic interactions critical to dimer stabiHzation. As a result, de-oligomerization occurs rapidly upon injection and the product can be administered at meal times rather than 30 min before. The different forms and formulations of insuHn are shown on the supplementary CD-ROM. 

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