Isolation, cloning and optimization for expression of neutrophil gelatinase associated lipocalin (NGAL) in prokaryote
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Abstract
Acute kidney injury (AKI) is a sudden loss of kidney function followed by a failure to maintain fluid, which is associated with increased mortality. Recently, neutrophil gelatinase associated lipocalin (NGAL) has been recommended as the first biomarker for prediction of kidney damage in the early phase. For medical applications of the NGAL protein, a large amount and expression system of this protein is required. Currently, the assay to determine NGAL levels is enzyme-linked immunosorbent assay (ELISA), which is accurate but inconvenient for clinical applications, because its long-time process takes about 4 hours and the antibody against human NGAL is expensive. Therefore, high-level expression of NGAL is required in order to develop a commercially NGAL strip test. The present study aimed to construct the recombinant plasmid, to optimize the expression of NGAL in Escherichia coli BL21(DE3) and OrigamiTMB(DE3) strains. The host strain, IPTG concentration and induction time were also evaluated. NGAL gene was cloned in pET32a and transformed to E. coli BL21(DE3) and OrigamiTMB(DE3). The construct of NGAL and the expression vector was successful. BL21(DE3) showed a high growth rate indicated by reaching the mid-log phase faster than OrigamiTMB(DE3). However, both E. coli BL21(DE3) and OrigamiTMB (DE3) showed no expression of NGAL. In conclusion, this study demonstrates the efficiency of the expression host to produce protein for development as a commercial strip test. The suitable system to express NGAL protein should be further investigated.
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