Purification of Recombinant Human Serum Albumin (rHSA) Produced by Genetically Modified Pichia Pastoris

• Recombinant human serum albumin (rHSA) was produced by genetically transformed Pichia pastoris yeast. The cell-culture supernatant (CCS) contained 8–12 g/l rHSA that was purified in a three-step procedureinvolving (1) a capture step using the newly developed cation exchanger CaptoTM MMC; (2) an intermediate step using Phenyl SepharoseTM and, (3) a polishingstep using Aminobutyl SepharoseTM 6 FF. The total recovery was 25–35% and the product fulfils the purity criteria of the European Pharmacopeia. Purified rHSA and plasma-derived HSA were essentially identical judging bySDS- or native-PAGE, and the pigment level (expressed as A 350/A280) in the rHSA was 0.03 or less and was strongly dependent on the quality of the CCS.Dimers and polymers in the final product were less than that found in purified plasma-derived HSA. The molar mass of the purified rHSA, as well as of its natural counterpart, is 67 000 Daltons by MALDI-ToF mass spectrometry, while the iso-electric points of both recombinant and natural HSA ranged between pH 5.42–5.55 when determined in 8M urea. The stability profiles of both proteins after heat treatment were identical as determined by differential scanning calorimetry (DSC). The results obtained here suggest the purified rHSA to be a homogeneous protein identical to its natural counterpart.

Ämnesord

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

Nyckelord

Fermentation

HSA

human serum albumin

P. pastoris

purification

rHSA

SDS-PAGE

Chemistry

Kemi

Biochemistry

biokemi

ytbioteknik

Surface Biotechnology

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