Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations.

To stabilize Aspergillus awamori glucoamylase (GA), three proline substitution mutations were constructed. When expressed in Saccharomyces cerevisiae, Ser30-->Pro (S30P) stabilized the enzyme without decreased activity, whereas Asp345-->Pro (D345P) did not significantly alter and Glu408-->Pro (E408P) greatly decreased enzyme thermostability. The S30P mutation was combined with two previously identified stabilizing mutations: Gly137-->Ala, and Asn20-->Cys/Ala27-->Cys (which creates a disulfide bond between positions 20 and 27). The combined mutants demonstrated cumulative stabilization as shown by decreased irreversible thermoinactivation rates between 65 and 80 degrees C. Additionally, two of the combined mutants outperformed wild-type GA in high-temperature (65 degrees C) saccharifications of DE 10 maltodextrin and were more active than the wild-type enzyme when assayed using maltose as substrate.

Characterization and polyelectrolyte precipitation of beta-galactosidase containing genetic fusions of charged polypeptides.

Genetically engineered versions of beta-galactosidase were constructed through the addition of charged polypeptide fusion tails for the purpose of enhancing polyelectrolyte precipitation. Negatively charged aspartic acid tails and positively charged poly(arginine) tails were added to beta-galactosidase from Escherichia coli. These fusion proteins were all shown to possess specific activity equal to that of the native enzyme. Gel permeation and ion-exchange chromatography provided evidence concerning the integrity of the tails as well as their altered charge characteristics. All enzymes containing charged tails displayed enhanced polyelectrolyte precipitation over the native enzyme. An optimal number of charged residues, beyond which no further enhancement of precipitation was observed, was found to be approximately 10 residues for each type of tail. No interference from nucleic acids was observed in the precipitation of positively tailed beta-galactosidase.

Frequency of long-term lower limb ischemia associated with intraaortic balloon pump use.

Lower limb ischemia is a frequent complication of intraaortic balloon pump (IABP) use. The incidence and risk factors for acute ischemia have been well-defined, but little is known about long-term ischemic complications. This prospective study evaluated the incidence, nature, progression and predisposing factors for long-term lower limb ischemia in 151 patients who were previously treated with the IABP. These persons were interviewed and their lower extremities examined 12 to 20 months after undergoing IABP counterpulsation. Limb ischemia, characterized primarily by ipsilateral discomfort and diminished pulses, occurred in 18% of those evaluated. Evidence of ischemia worsened over time in 14%. Logistic regression analysis, which was based on variables found to be significant in bivariate analysis, revealed that the occurrence of limb ischemia acutely, cardiogenic shock as an indication for IABP insertion, and smoking (at the time of hospitalization or having quit < 10 years previously) were risk factors for long-term lower limb ischemia. The adjusted odds ratio for acute limb ischemia was 8.89 (95% confidence interval 2.80 to 28.21), for cardiogenic shock 3.59 (95% confidence interval 1.01 to 12.75), and for smoking 2.87 (95% confidence interval 1.10 to 7.46). Increasing numbers of patients are undergoing IABP counterpulsation and a greater proportion of these are surviving their acute event and resuming active lives. It is essential to recognize that detrimental consequences of this device can persist long after hospitalization.

A restriction fragment length polymorphism map and electrophoretic karyotype of the fungal maize pathogen Cochliobolus heterostrophus.

A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical length to map distance should make this RFLP map a useful tool for cloning genes.

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.

Characterization of the soluble human granulocyte-macrophage colony-stimulating factor receptor complex.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-R) is expressed on both hematopoietic and non-hematopoietic tissues. Although the receptor has been identified by cross-linking studies as an 84,000-dalton protein, very little is known about its biochemistry. In this report, we describe a soluble binding assay for the human GM-R which allowed us to characterize the receptor complex from various sources, including plasma membranes of placenta, neutrophils, and human myeloid leukemia cell lines. Preparation of membranes as well as solubilization by Triton X-100 and N-octylglucoside resulted in a 5-10-fold lower affinity of the receptor for GM-CSF. The Kd decreased from 20 to 80 pM in intact cells to 200-500 pM in both intact and solubilized membranes. Binding in solution was rapid, specific for GM-CSF, and best fit a "one-site" model with an approximate Kd of 500 pM. The dissociation rate constant for the soluble GM-R was very similar to that of intact cells (k2 = 0.013 min-1 versus 0.017 min-1, respectively). As expected, solubilized membranes obtained from those cells expressing the highest number of GM-R (neutrophils and dimethyl sulfoxide-induced HL-60 cells; approximately 500-800 sites/cell) possessed the highest concentration of soluble GM-R (approximately 2-3 x 10(8) GM-R/micrograms). Cross-linking of 125I-GM-CSF to soluble GM-R resulted in the appearance of two specifically labeled complexes. A major 110-kDa receptor-ligand complex is found when cross-linking is performed with intact cells; both 110- and 200-kDa species are seen when cross-linking is performed with either intact membranes or soluble GM-R. These studies define methods by which intact GM-R can be solubilized and measured in solution, permitting a more complete biochemical characterization of the intact GM-R complex.

Recovery of a charged-fusion protein from cell extracts by polyelectrolyte precipitation.

Beta-galactosidase served as a model system to explore the feasibility of enhancing the selectivity of a low-cost, easily scaled separation method-precipitation. Enhanced selectivity was sought by fusing the enzyme with polypeptide tails including 5 and 11 aspartates. The unfused protein could not be selectively removed from the Escherichia coli cell extract by precipitation with polyethylenimine (PEI), but the longest fusion could be selectively removed. The presence of nucleic acids limited the purification attainable. Pretreatment with nuclease followed by diafiltration resulted in an extract from which the same fusion could be precipitated with greater than fivefold enrichment, while the untailed enzyme remained unenriched by the same precipitation step. Selectivity is attributed to the binding strength of the polyanionic tails to the polycationic PEI.

Polyelectrolyte precipitation of beta-galactosidase fusions containing poly-aspartic acid tails.

Protein recovery from industrial microbial processes can be very expensive, often exceeding the cost of protein production. We have genetically engineered 3 beta-galactosidase (beta-gal) fusion proteins containing poly-aspartic acid tails to test the effect of the tails on recovery by the relatively inexpensive method of polyelectrolyte precipitation. The fusion proteins, designated T1, T2, and T3, were constructed with C-terminal tails of 5, 11, and 16 aspartic acid residues, respectively. The fusion proteins were expressed in Escherichia coli, and purified by affinity chromatography. T1 and T2 had specific activities similar to that of wildtype beta-gal, whereas the specific activity of T3 was about half that of T1 and T2. The increased net charge of the fusion proteins compared to wildtype beta-gal was indicated both by ion-exchange chromatography and their migration pattern in non-denaturing polyacrylamide gel electrophoresis. All three tails enhanced polyethyleneimine (PEI) precipitation of the fusion proteins compared to wildtype beta-gal. At a low PEI/protein ratio (0.01, g g-1), recovery by precipitation of T2 and T3 was more than 2 X that of the beta-gal control, whereas that of T1 was only slightly greater than that of the control. At a higher PEI/protein ratio (0.03, g g-1) the amount of precipitation of all three fusion proteins was nearly the same, about 1.5 X that of the control.

Use of an oligonucleotide probe to detect transplacement of an amber mutation into a yeast histone H3 gene.

We have used a synthetic 17-mer to direct mutagenesis of the cloned yeast histone H3 gene HHT2, creating an amber mutation at amino acid 41. This point mutation did not alter the restriction pattern of the HHT2 gene nor was it expected to provide an easily scorable phenotype in vivo. Therefore, nucleic acid hybridization was used to detect this point mutation during strain construction. The oligonucleotide was used to probe yeast genomic Southern blots to detect integration of the plasmid bearing the mutant HHT2 gene into the genome, and then to score the eventual excision of the plasmid vector with retention of the mutant gene on the chromosome. This technique can be used to score virtually any engineered point mutations in yeast.