Human serum albumin (HSA) is a plasma protein with a molecular weight of 66.5 kDa, which is most widely used in the pharmaceutical and clinical fields. One application of HSA in a drug delivery system is to increase the relaxation time on magnetic resonance imaging. The combination of contrast agent, gadolinium-diethylenetriamine pentaacetate (Gd-DTPA), with HSA can produce better imaging. This study aims to express the recombinant HSA (rHSA) in the Pichia pastoris protease-deficient host, SMD1168, purification using chromatographic techniques and conjugation with Gd-DTPA. Optimization of rHSA expression by P. pastoris was done by varying the methanol inducer, 0.75%, 1.0%, 1.25%, and 1.5%, as well as characterization by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, the rHSA was purified using gel filtration, characterization by Size Exclusion Chromatography- High Performance Liquid Chromatography (SEC-HPLC), and conjugation with Gd-DTPA. The rHSA (~66.5 kDa) was characterized from broth medium of P. pastoris SMD1168. The optimum conditions for rHSA expression were 1.5% methanol at 48 hours incubation, which resulted in 0.44 g/l of rHSA. The rHSA with 81.84% purity was obtained by gel filtration, and conjugation with Gd-DTPA produced high-purity Gd-DTPA-rHSA, demonstrated by a single peak (rt = 10.493 minutes). The protease-deficient P. pastoris was successfully used to express intact rHSA and the rHSA was successfully conjugated to Gd-DTPA with high purity.
Key words: recombinant Human Serum Albumin, P. pastoris protease-deficient host, Gadolinium-Diethylene Triamine Penta Acetate-HSA.
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