Facilitated downstream processing of a histidine-tagged protein from unclarifiedE. coli homogenates using immobilized metal affinity expanded-bed adsorption

Abstract

The facilitated downstream processing of an intracellular, polyhistidine‐tagged protein, glutathione S‐transferase [GST‐(His)6], direct from unclarified E. coli homogenates using expanded beds of STREAMLINE chelating, has been investigated. A series of pilot experiments were used to develop preparative‐scale separations of GST‐(His)6, initially in packed and then in expanded beds. Packed beds of Ni2+‐loaded STREAMLINE chelating proved to have the highest 5% dynamic capacity for GST‐(His)6, of 357 U mL−1 (36 mg mL−1). When using immobilized Cu2+ or Zn2+, metal ion transfer was observed from the iminodiacetate ligands to the high‐affinity chelator, GST‐(His)6. The subsequent metal affinity precipitation of this homodimer resulted in operational problems. An equilibrium adsorption isotherm demonstrated the high affinity of GST‐(His)6 for immobilized Ni2+, with a qm of 695 U mL−1 (70 mg mL−1) and a Kd of 0.089 U mL−1 (0.0089 mg mL−1). Ni2+‐loaded STREAMLINE chelating was therefore selected to purify GST‐(His)6 from unclarified E. coli homogenate, resulting in an eluted yield of 80% and a 3.34‐fold purification. The high dynamic capacity in the expanded mode of 357 U mL−1 (36 mg mL−1) demonstrates that this specific interaction was not affected by the presence of E. coli cell debris. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 206–216, 2000.

Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas

“…[12][13][14]24 Além disso, a cauda é pouco imunogênica, não carregada em pH fisiológico e raramente interfere na estrutura ou função da proteína alvo. 3,4,25,26 Finalizada a etapa de purificação, caso seja necessário, a ligação entre a cauda e a proteína alvo pode ser rompida empregandose métodos químicos ou enzimáticos que preservem a estrutura e a atividade da proteína de interesse.…”

“…Na cromatografia em leito expandido, um fluxo ascendente de fase móvel na coluna promove a expansão do leito -distanciamento das partículas de adsorvente (géis especiais, por exemplo, Streamline Chelating ® , Amersham Pharmacia) de velocidade terminal diferente, devido à força de arraste -que, desta forma, pode receber alimentação com particulados sem que haja colmatação (entupimento) do leito. 25 Na cromatografia em membranas, o transporte das moléculas da solução até o interior dos poros é regido, sobretudo, pela convecção e não pela difusão (como ocorre nos géis). Este transporte convectivo facilita o acesso da biomolécula ao sítio de fixação do ligante.…”

A Study of the Interaction of HEK-293 Cells with Streamline Chelating Adsorbent in Expanded Bed Operation

“…The affinity depends on the presence of certain surface exposed amino acid residues such as histidine, tryptophan, and cysteine (12). The use of IMAC in an EBA column has been evaluated for the direct recovery of proteins from yeast cells (13), mammalian cells (5), bacterial cells (14)(15)(16)(17)(18)(19), and human plasma (20). Streamline Chelating is an IMAC adsorbent designed for EBA, and it also serves as a weak cation exchange adsorbent when no metal ion is primed to its iminodiacetic acid (IDA) ligand.…”

Separation of Tricomponent Protein Mixtures with Triblock Nanorods

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] A number of generic affinity-based purification methods for recombinant proteins have been developed by proteomic researchers. [7][8][9][10][11][12][13][14][15][16][17][18] These methods are based upon the use of specific antibodies or affinity tags that are covalently attached to the protein of interest.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] A number of generic affinity-based purification methods for recombinant proteins have been developed by proteomic researchers. [7][8][9][10][11][12][13][14][15][16][17][18] These methods are based upon the use of specific antibodies or affinity tags that are covalently attached to the protein of interest. Usually short peptides with specific molecular recognition properties such as a maltose binding protein, 8 streptag, 9 cellulose binding domain, 10 glutathione S-transferase, 11 polyhistidine, [12][13][14] and biotin [15][16][17][18][19][20] are used as such tags.…”