Efficient and easily scalable protein folding strong anion exchange chromatography for renaturation and simultaneous purification of recombinant human asparaginase from E. coli

• Published in: Biotechnology progress Vol. 34; no. 4; pp. 1036 - 1044
• Main Authors: Kante, Rajesh Kumar, Vemula, Sandeep, Mallu, Maheswara Reddy, Ronda, Srinivasa Reddy
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.07.2018
• Subjects: Additives; Anion exchange; anion exchange chromatography; Anion Exchange Resins - chemistry; Anion exchanging; Arginine; Asparaginase; Asparaginase - chemistry; Asparaginase - isolation & purification; Chromatography; Chromatography, Ion Exchange - methods; Dextran; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Folding; Fructose; Glycerol; Humans; Inclusion bodies; Organic chemistry; pH effects; Phase composition; Protein Folding; protein folding liquid chromatography; Protein purification; Proteins; Purification; Purity; purity, recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recovery; recovery yield; Renaturation; Sorbitol; specific activity; Streaming; protein folding liquid chromatography; anion exchange chromatography; recovery yield; specific activity; purity, recombinant proteins
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1002/btpr.2649

Recombinant proteins are revolutionizing present day therapeutics. They are generally expressed as insoluble inclusion bodies in the E. coli and mis‐folding, loss of protein, and high cost of down streaming are the hurdles in their recovery. For the first time, we are reporting the refolding with simultaneous purification of rhASP in E. coli using a single step utilizing protein folding‐strong anion exchange chromatography (PF‐SAX). The purification method is also standardized for optimal concentration of solution additives, pH, and mobile phase composition. The results showed purification of rhASP with anion exchange chromatography was effective. Phosphate buffer and slightly alkaline pH produced significant recovery yields and purity profiles. The effect of solution additives such as arginine, glycerol, TMAO, sorbitol, dextran, glutamate, and fructose on rhASP renaturation is also investigated. Significant results were achieved using arginine‐TMAO combination in terms of purity, recovery yield and specific activity of 99%, 78%, and 210 IU/mg, respectively. The work concludes that PF‐SAX refolding method is superior to other conventional methods and it can be applied to large scale purification of rhASP produced in E. coli. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1036–1044, 2018