Tolerance of Escherichia coli beta-galactosidase C-terminus to different-sized fusions.

The tolerance of the beta-galactosidase C-terminus to foreign protein fusions has been explored by using different-sized derivatives of the chimeric protein LACVP1. While the molecular mass of the partner domain shows a minor influence on protein toxicity for the producing E. coli cells, it dramatically affects the proteolytic susceptibility of the whole fusion. Surprisingly, the observed structural modulation of proteolysis is not an all-or-nothing process, but it exhibits a continuous effect concomitantly with the length of the fusion. The conformational effects caused by increasingly sized partners seem to progressively expose cryptic protease target sites, initiating a proteolytic cascade that dramatically reduces the yield of the recombinant protein.     

Ion exchange immobilization of changed beta-galactosidase fusions for lactose hydrolysis

The use of charged peptides fused to enzymes for immobilization onto ion-exchange membranes was explored for the enzyme x-galactosidase. The additional charged peptides, containing 1, 5, 11, and 16 aspartates, fused to x-galactosidase, for the most part did not interfere with the kinetic behavior for lactose hydrolysis. There was a 2-fold decline in V(m) for the 16-aspartate fusion, but the others were quite similar to the wild type enzyme (BGWT). BGWT and the fusions all retained approximately 50% of their activities when adsorbed onto ion-exchange membranes. In contrast to BGWT, the enhanced binding strength of the 11 aspartate fusion provided the ability to hydrolyze whey permeate at 0.3 M ionic strength without enzyme leakage, and to immobilize the enzyme directly from diluted cell extract with 83% purity. (c) 1994 John Wiley & Sons, Inc.     

Isolation of fla-lacZ fusions in Escherichia coli K-12: most fusions result in soluble beta-galactosidase

A series of fusions of flagellar genes to the lacZ gene was generated by insertion of Mu dII301 (Apr lac) bacteriophage into the genome of Escherichia coli. The beta-galactosidase activity in each resulting mutant was measured, and the location of the activity in the membrane, periplasmic, or cytoplasmic fraction of the cell was determined. There were three classes of mutants: those which had beta-galactosidase activity mainly in the membrane fraction, those which had it distributed in the soluble and membrane fractions, and those which had it in the cytoplasmic fraction only. The last, soluble-fraction-only, class was predominant in fla-lac gene fusions. In particular, the following mutants were shown to have beta-galactosidase activity in the membrane fractions: on the inner membrane, mutants with flaB fusions, and on the inner and outer membranes, mutants with flaA4850, flaM, and flaU4849 fusions. These results suggest that fla-lacZ gene fusions produce proteins which are able to detect the signals of the leader sequence and the membrane-anchoring region of the flagellar system.     

Polyelectrolyte precipitation of proteins: I. The effect of reactor conditions

Lysozyme was recovered from egg white by continuous precipitation with polyacrylic acid (molecular weight of 4 x 10(6)). Precipitator residence time and shear rate had significant effects on the size distribution of the precipitate, but no clear effects on the compositions. Precipitate mean size increased with higher shear, indicating growth phenomena predominating over breakage. Also, an enhancement of growth rate at small sizes was noted. The Camp number successfully characterized the interaction of shear rate and residence time on the particle size.     

Polyelectrolyte precipitation of proteins: II. Models of the particle size distributions

A population-balance model has been used to characterize continuous polyelectrolyte precipitation of egg white proteins. We have modeled the particle size distributions of aggregates formed under a range of mixing conditions. The models, accounting for aggregate growth (by both shear-driven and Brownian-like collisions), breakage (by hydrodynamic shear or aggregate-aggregate collisions), and birth (by the breakage of large aggregates), fit the data well. The kinetic constants show dependencies on shear rate and residence time that have not been previously theoretically predicted; these dependencies are due in part to aging effects on the aggregate. The model constants show a dominance of growth over breakage, supporting qualitative interpretations of the particle size distributions. A mechanism for growth-rate enhancement, caused by polymer extensions from the particle surfaces, produced improved model performance. A collisional breakage mechanism is supported.     

Primary sequence of the EcoRII endonuclease and properties of its fusions with beta-galactosidase

The EcoRII endonuclease cleaves DNA containing the sequence CC(A/T)GG before the first cytosine. The methylation of the second cytosine in the sequence by either the EcoRII methylase or Dcm, a chromosomally coded protein in Escherichia coli, inhibits the cleavage. The gene for the EcoRII endonuclease was mapped by analysis of derivatives containing linker insertions, transposon insertions, and restriction fragment deletions. Surprisingly, plasmids carrying the wild-type endonuclease gene and the EcoRII methylase gene interrupted by transposon insertions appeared to be lethal to dcm+ strains of E. coli. We conclude that not all the EcoRII/Dcm recognition sites in the cellular DNA are methylated in dcm+ strains. The DNA sequence of a 1650-base pair fragment containing the endonuclease gene was determined. It revealed an open reading frame that could code for a 45.6-kDa protein. This predicted size is consistent with the known size of the endonuclease monomer (44 kDa). The endonuclease and methylase genes appear to be transcribed convergently from separate promoters. The reading frame of the endonuclease gene was confirmed at three points by generating random protein fusions between the endonuclease and beta-galactosidase, followed by an analysis of the sequence at the junctions. One of these fusions is missing 18 COOH-terminal amino acids of the endonuclease but still displays significant ability to restrict incoming phage in addition to beta-galactosidase activity. No striking similarity between the sequence of the endonuclease and any other protein in the PIR data base was found. The knowledge of the primary sequence of the endonuclease and the availability of the various constructs involving its gene should be helpful in the study of the interaction of the enzyme with its substrate DNA.     

Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails

Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn(2+). The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity. (c) 1996 John Wiley & Sons, Inc.     

N-terminal amino acid sequence of persimmon fruit beta-galactosidase.

beta-Galactosidase (EC 3.2.1.23) from persimmon fruit was purified 114-fold with a 15% yield using Sephadex G-100 gel filtration, CM-Sephadex ion exchange, and Sephacryl S-200 gel filtration chromatography, with subsequent electroelution from nondenaturing polyacrylamide gel electrophoresis (PAGE) gels. The estimated molecular mass of the native beta-galactosidase by Sephacryl S-200 was 118 kD. After sodium dodecyl sulfate-PAGE of the enzyme electroeluted from native gels, two subunits with estimated molecular masses of 34 and 44 kD were observed, suggesting that the native enzyme was an aggregate of several subunits. Amino acid composition and N-terminal amino acid sequences of the two major subunits were different.     

Metal affinity precipitation of proteins carrying genetically attached polyhistidine affinity tails

In this study, galactose dehydrogenase (EC 1.1.1.48) was chosen as a prototype target protein to investigate the capability of metal affinity precipitation to facilitate the purification of genetically engineered proteins. A DNA fragment encoding five histidine residues was fused to the 3'-terminal end of the galactose dehydrogenase gene from Pseudomonas fluorescens and thereafter expressed in Escherichia coli. The additional five histidines functioned as an affinity tail and the modified enzyme could be purified using metal affinity precipitation when the metal-chelate complex with ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetra-acetic acid, EGTA(Zn)2, was added to the protein solution. The affinity tail could also be applied for the purification of the fusion protein utilising immobilised metal affinity chromatography. After purification, the pentahistidine affinity tail could be removed enzymatically by carboxypeptidase A. Furthermore, growth rate experiments demonstrated that the expression of the metal-binding affinity tail in E. coli cells enhanced the tolerance to zinc ions when added to the growth medium.     

Improved system for construction and analysis of single-copy beta-galactosidase operon fusions in Yersinia enterocolitica

We report a significantly improved system for studying single-copy lacZ operon fusions in Yersinia enterocolitica: a simple procedure for the stable integration of lacZ operon fusions into the ara locus and a strain with a deletion mutation that abolishes the low level of endogenous beta-galactosidase activity.     

Heterogeneity of acid beta-galactosidase from rabbit kidney

1. Rabbit kidney acid beta-galactosidase can be resolved into three peaks (named A3, A2 and A1) by gel-filtration chromatography. Their estimated molecular weights were: more than 250,000, 150,000 and 17,000 respectively. 2. The purified acid form appeared as a single band of protein (Mr = 28,000) on electrophoresis in the presence of sodium dodecyl sulphate, suggesting that forms A3 and A2 are multimeric forms of beta-galactosidase A1. 3. Treatment with neuraminidase from Clostridium perfringens converts form A3 into a more basic form. This phenomenon occurs also when this form is stored for a week at 4 degrees C and parallels its disaggregation. 4. The data suggest that the sialic acids present in the multimeric forms are involved in the aggregation of the acidic form of beta-galactosidase.     

Observations on the formation of clones containing araB-lacZ cistron fusions

Summary Casadaban (1976) developed a technique for isolating E. coli clones containing fusions of the amino terminal-encoding portion of any cistron with the carboxy terminal-encoding portion of lacZ. The technique utilizes prophage Mu homology to bring the two cistrons into proximity. I have followed the appearance over time of colonies containing araB-lacZ fusions from a strain where the begining of the araB cistron is connected to lacZ by an intact Mucts62 prophage. Cultures of the starting strain grown on a variety of media have fewer than 2 in 10¹⁰ cells capable of forming colonies within three days after plating on selective arabinose-lactose medium. At 32°C, there is a delay of between 4 and 19 days before the first colony appears. The kinetics of colony appearance over the next two to four weeks then shows a rapid increase in the number of new colonies emerging per day followed by a decline. The pattern of colonial emergence and the final numbers of fusion colonies obtained are not grossly affected by reducing the number of cells plated over five orders of magnitude. Fusion colonies sometimes show a clustered pattern when they first emerge. Innoculation of pre-existing fusion clones at specific locations on the arabinose-lacteredselection plates seeded with the starting strain leads to the formation of inhibitory zones where no fusion colonies appear. Selection plates contain many microcolonies and papillae which do not proliferate into scoreable colonies but nonetheless contain cells capable of growth when replated on the same selective medium. Up to 39% of all plated cells are capable of producing fusion clones. The kinetics of fusion colony appearance can be altered by environmental and genetic manipulations. Partial derepression of the Mucts prophage at 37° accelerates the appearance of colonies but also reduces the final yield. Addition of limiting concentrations of glucose to the selective medium also accelerates the appearance of colonies in a specific fashion: enrichments below the level required for maximum acceleration produce a biphasic kinetics with two waves of fusion clone emergence separated by an eight-day interval. Infection with Muc ⁺ pAp phage produces dilysogens that have almost completely lost the ability to produce fusions. Infection with MuctsAampAP phage produces strains that are reduced in phage production and have delayed kinetics of fusion clone emergence. The implications of these observations for theories of hereditary change in bacteria are discussed.     

The precipitation of poly-L-glutamic acid. II. beta-Precipitation.

Infrared and X-ray diffraction studies have established that in the β-precipitation region of poly-L -glutamic acid the chains are in the β-conformation. Therefore, a major molecular conformational change has taken place upon precipitation. It is shown that the size of the α-helical aggregates remains constant with time in the β-region. Strong evidence can be offered to indicate that the transformation involves a transitory random-coil intermediate. Reasons are advanced, in view of the stability of the β-form, as to why two distinct precipitation regions exist.     

The precipitation of poly-L-glutamic acid. I. Alpha-precipitation.

Studies have been made of the precipitation of poly-L -glutamic acid from dilute aqueous solutions, over the pH range where the α-helical conformation is maintained. The major purpose is to establish the molecular conformation in the precipitate and to outline the mechanism of the precipitation. Utilizing molecular-weight fractions, it is shown that there are two distinctly different precipitation regions, which depend on temperature, concentration, and pH. In one of these regions, the α-region, a variety of physical properties demonstrate that precipitation occurs without any conformational change. The temperature coefficient of the precipitation process in this region indicates that it is nucleation-controlled.     

Charged fusions for selective recovery of beta-galactosidase from cell extract using hollow fiber ion-exchange membrane adsorption

We explored the use of charged fusions for selective recovery of beta-galactosidase from cell extract using a low-cost, easily scaled, fast, charge-based separation technique-ion exchange on hollow fiber ion-exchange membranes (HFIEMs). The additional charges carried by a series of anionic fusion tails allowed selective binding and release of beta-galactosidase from Escherichia coli cell extract using the HFIEM cartridge. The purification factors increased with fusion length. The beta-galactosidase was recovered in active form. For the longest fusion studied, more than sixfold enrichment in specific activity was attained. The specific activity of the recovered fraction is comparable with that of commercial wild-type beta-galactosidase and affinity-purified fusion protein. (c) 1993 John Wiley & Sons, Inc.     

Amino acid sequence of beta-galactosidase. V. Isolation and sequences of chymotryptic peptides.

The amino acid sequence of 72 chymotryptic peptides isolated from 14C-, 3H-labeled carboxymethyl-beta-galactosidase has been determined. A variety of techniques were used in the isolation procedures including separation by solubility, size, and ion exchange and paper chromatography. These peptides contain approximatley 500 amino acids, range in size from 2 to 26 residues, and give overlaps with tryptic peptides of 16 to 55 residues. Peptides from this digest and those reported earlier from tryptic digests account together for the sequence of about 600 of the 1021 residues in the subunit.