Ilari Suominen

Fusion tails for the recovery and purification of recombinant proteins

Several fusion tail systems have been developed to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Tails with a variety of characteristics have been used: (1) entire enzymes with affinity for immobilized substrates or inhibitors; (2) peptide-binding proteins with affinity to immunoglobulin G or albumin; (3) carbohydrate-binding proteins or domains; (4) a biotin-binding domain for in vivo biotination promoting affinity of the fusion protein to avidin or streptavidin; (5) antigenic epitopes with affinity to immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) poly(His) residues for recovery by immobilized metal affinity chromatography; and (8) other poly(amino acid)s, with binding specificities based on properties of the amino acid side chain. Fusion tails are useful at the lab scale and have potential for enhancing recovery using economical recovery methods that are easily scaled up for industrial downstream processing. Fusion tails can be used to promote secretion of target proteins and can also provide useful assay tags based on enzymatic activity or antibody binding. Many fusion tails do not interfere with the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of authentic proteins a site for specific cleavage is often included, allowing removal of the tail after recovery.

Extracellular production of cloned α-amylase by Escherichia coli

Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.

Oxidation of Crocein Orange G by lignin peroxidase isoenzymes. Kinetics and effect of H2O2.

The ligninolytic enzyme system ofPhanerochaete chrysosporiun is able to decolorize several recalcitrant dyes. Three lignin peroxidase isoenzymes, LiP 3.85, LiP 4.15, and LiP 4.65, were purified by preparative isoelectric focusing from the carbon-limited culture medium ofP. chrysosporium. Based on amino terminal sequences, the purified isoenzymes correspond to the isoenzymes H8, H6, and H2, respectively, from theN-limited culture. The purified isoenzymes were used for decolorization of an azo dye, Crocein Orange G (COG). According to the kinetic data obtained, the oxidation of COG by lignin peroxidase appeared to follow Michaelis-Menten kinetics. Kinetic parameters for each isoenzyme were determined. The inactivating effect of ascending H2O2 concentrations on COG oxidation is shown to be exponential within the used concentration range. The best degree of decolorization of 100 μM COG was obtained when the H2O2 concentration was 150 μM. This was also the lowest H2O2 concentration for maximal decolorization of 100 μM COG, regardless of the amount of lignin peroxidase used in the reaction.

Enhanced recovery and purification of Aspergillus glucoamylase from Saccharomyces cerevisiae by the addition of poly(aspartic acid) tails

Poly(aspartic acid) tails of different lengths were fused to the glucoamylase (GA) of Aspergillus awamori by genetic engineering techniques. Tails consisting of 5, 7, and 10 aspartate residues were fused to the N-terminus of the full-length mature GA (aa 1-616) downstream from the intact leader peptide to produce fusion proteins designated GAND5, GAND7, and GAND10, respectively. Three fusion proteins with C-terminal tails were also constructed, designated GACD0, GACD5, and GACD10 (0, 5, and 10 aspartate residues, respectively). For the C-terminal fusion proteins, the tails were fused to a catalytically active but truncated form of GA (aa 1-484). All of the charged tails had the general sequence Met-Ala-Aspn-Tyr, where n = 0, 5, 7, or 10. The modified genes were expressed in the yeast Saccharomyces cerevisiae and the proteins secreted into the culture medium. The enzymes were subsequently purified by affinity chromatography. The specific activity of each purified enzyme was found to be comparable to the wild-type enzyme. The C-terminal tails did not interfere with expression, whereas decreased extracellular glucoamylase activities corresponding to increased tail length were found for the N-terminal fusion proteins. Amino-terminal amino acid sequence analysis of the purified GAND proteins confirmed the authenticity of the amino termini of the modified proteins and showed that both the leader peptidase and KEX2 protease cleavages had occurred faithfully. The increased net negative charge of the GAND and GACD proteins was indicated by both nondenaturing PAGE and isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of signal peptide mutations on processing of Bacillus stearothermophilus α-amylase in Escherichia coli

Bacillus stearothermophilus alpha-amylase has a signal peptide typical for proteins exported by Gram-positive bacteria. There is only one signal peptidase processing site when the protein is exported from the original host, but when it is exported by Escherichia coli, two alternative sites are utilized. Site-directed mutagenesis was used to study the processing in E. coli. Processing sites for 13 B. stearothermophilus alpha-amylases carrying mutations in their signal peptide were determined. Processing of the signal peptide was remarkably tolerant to mutations, because switching between the alternative sites was possible. The length and the sequence of the region between the hydrophobic core and the cleavage site was crucial for determining the choice of the processing site. Some mutations more distal to the cleavage site also affected the site preference.

Thermostable Alpha Amylase of Bacillus Stearothermophilus: Cloning, Expression, and Secretion by Escherichia Coli

The gene coding for a thermostable extracellular α-amylase was cloned from Bacillus stearothermophilus in Escherichia coli using lambda EMBL3 vector. The gene was subcloned in plasmids pUC-8 and pBR322. Nucleotide sequence of the gene was determined and it was shown to contain an open reading frame of 1650 bp coding for a preprotein of MW 63,000. The enzyme has a typical signal sequence for protein secretion. Processing of the signal peptide is ambiguous in E. coli occurring at two sites separated by three amino acid residues. The mature enzyme is homologous with two other liquefying α-amylases from B. amyloliquefaciens and B. licheniformis. The enzyme is initially secreted into the periplasm of E. coli, but when sufficient amount of α-amylase has accumulated in the stationary phase of growth, it is also found in the culture medium.

Phage lambda pL promoter controlled α-amylase expression inEscherichia coli during fermentation

SummaryPhage lambda pL promoter controlled expression of theBacillus stearothermophilus gene coding for a thermostable α-amylase inE. coli was studied in shake flask cultures and in a laboratory fermenter. At an inducible temperature (40 °C) the final cell density was lower, but the total enzyme activity produced ca. 80% higher than at a non-inducible temperature (30 °C). Moreover, 17% of the total enzyme activity was found in the culture medium. The α-amylase yield, production rate and proportion secreted were further increased by shifting the fermentation temperature after certain period of bacterial growth rather than at the beginning of fermentation.