Expression stability maturation

The expansion of our knowledge of the structure and function of Na,K-ATPase is reflected in a rapid succession of reviews on Na,K-ATPase genes and regulation of expression [17], subunit assembly and functional maturation [20], the isozymes of Na,K-ATPase [18], and the stability of a subunit isoforms during evolution [21], physiological aspects and regulation of Na,K-ATPase [22], reconstitution and cation exchange [23], chemical modification [24], and occlusion of cations [25]. Other valuable sources are the review articles [26] and recent developments [27] reported at the International Na,K-pump Conference in September 1990. 

In an Escherichia coli expression system for the aqualysin I precursor, the precursor is processed autoproteolytically into the mature 28-kDa enzyme by treatment at 65 ° C.23) In this case, the N-terminal pro-sequence is required for the production of active enzyme and functions to stabilize the precursor structure.283 The C-terminal pro-sequence is not essential for the production of active aqualysin 1,293 but seems to be involved in the translocation of the precursor across the cytoplasmic membrane.303 In a Thermus thermophilus expression system,313 the C-terminal pro-sequence is required for the production and extracellular secretion of active aqualysin I.323 In an E. coli expression system for the subtilisin E gene, the N-terminal pro-sequence is essential for the production of active enzyme,333 as in the case of aqualysin I. The requirement of the pro-sequence is also shown in vitro for the refolding of the inactive mature protein to an active enzyme.34 353 The functions of the N-terminal pro-sequences of aqualysin I and subtilisin E seem to be similar. 

Increased thermal stability of proteins used for biosensors is desirable to allow for robust devices that can withstand a variety of storage, assay, and regeneration conditions. In addition, some evidence suggests that starting from the most stable version of a bioreceptor by yeast surface display will aid in later affinity maturation efforts (38-40). In addition, thermally stable mutants can enable higher expression levels as soluble proteins from yeast or E. coli (19, 20, 38). Thermal stability selection rounds have been carried out on many of the proteins that were later mutated for high affinity (17,19-21, 38 2). 

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