title: Cell surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO₂ sequestration.
abstract: Carbonic anhydrase (CA) has recently gained renewed interests for its potential as a mass-transfer facilitator for CO(2) sequestration. However, the low stability and high price severely limit its applications. In this work, the expression of α-CA from Helicobacter pylori on the outer membrane of Escherichia coli using a surface-anchoring system derived from ice nucleation protein (INP) from Pseudomonas syringae was developed. To find the best surface anchoring motif, full-length INP (114 kDa), truncated INP (INP-NC, 33 kDa), and INP's N-domain with first two subunits (INP-N, 22 kDa) were evaluated. Two vectors, pKK223-3 and pET22b(+), with different promoters (T7 and Tac) were used to construct the fusion genes, and for each vector, three recombinant strains, each expressing a different length of the fusion protein, were obtained. SDS-PAGE, Western blot, immunofluorescence microscopy, FACS, and whole-cell ELISA confirmed the expression of fusion proteins on the surface of E. coli. The smallest fusion protein with INP-N as the anchoring motif had the highest expression level and CA activity, suggesting that INP-N is the best carrying protein due to its smaller size. Also, the T7 promoter in pET22b(+) induced with 0.2 mM IPTG gave high protein expression levels, whereas the Tac promoter in pKK223-3 gave low expression levels. The surface displayed CA was at least twofold more stable than that of the free form, and did not show any adverse effect on cell growth and outer membrane integrity. Cells with surface displayed CA were successfully used to facilitate CO(2) sequestration in contained liquid membrane (CLM).
authors: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio 43210, USA.
authors: Li-Hai
authors: LH
authors: Fan
authors: Ning
authors: N
authors: Liu
authors: Ming-Rui
authors: MR
authors: Yu
authors: Shang-Tian
authors: ST
authors: Yang
authors: Huan-Lin
authors: HL
authors: Chen
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002245:Carbon Dioxide / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D016480:Helicobacter pylori / Q000201:enzymology / Q000235:genetics; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D010957:Plasmids; D011401:Promoter Regions, Genetic; D044224:Pseudomonas syringae / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins; C063113:ice nucleation protein; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases