Application of vortex flow adsorption technology to intein-mediated recovery of recombinant human alpha1-antitrypsin.
Vortex flow is a secondary flow pattern that appears above a critical rotation rate in the annular gap between an inner rotating solid cylinder and an outer stationary cylindrical shell. By suspending adsorbent resin in the vortices, a novel unit operation, vortex flow adsorption (VFA), is created. In VFA, the rotation of the inner cylinder facilitates the fluidization of the adsorbent resin. Similar to expanded bed processes, VFA has high fluid voidage so that it can be used to recover biochemical products directly from fermentation broths or cell homogenates without removing cells or cell debris first. In this study, recombinant human alpha1-antitrypsin (alpha1-AT) was expressed in Escherichia coli as a fusion with a modified intein containing a chitin-binding domain. Therefore, the fusion protein can be recovered by chitin resin affinity adsorption. The intein can be induced to undergo in vitro peptide bond cleavage to specifically release alpha1-AT from the bound fusion protein. The capture efficiency of the fusion protein, 26.2%, was obtained in the VFA process. In addition, the specific activity of alpha1-AT was dramatically improved from 0.3 to 205.2 EIC/(mg total protein) after adsorption and cleavage. Therefore, vortex flow adsorption is an integrative technology to combine the primary clarification, concentration, and purification steps in conventional downstream processing into a single unit operation to efficiently recover and purify biochemical products.
Ma
Junfen
J
Massachusetts Institute of Technology, Department of Chemical Engineering, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. jfma@mit.edu
Cooney
Charles L
CL
D000327:Adsorption; D002686:Chitin / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012212:Rheology / Q000295:instrumentation / Q000379:methods*; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin; D002686:Chitin