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J M McCoy

Publications and source records attributed to J M McCoy.

At least 37 records · Page 2Linked to original sources
PubMed1995

Gene fusion expression systems in Escherichia coli.

In recent years, Escherichia coli gene fusion expression systems have circumvented many of the problems inherent in the use of this bacterium for the production of recombinant proteins. These systems also provide a powerful means for identifying peptides or proteins with desired binding specificities. Gene fusion technology continues to expand with the introduction of new fusion partners, purification and detection tags, cleavage reagents and ways to display peptides on the surface of bacteria.

Cloning, Molecular↗
PubMed1995

Expression of thioredoxin random peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: a system designed for exploring protein-protein interactions.

We have developed a system for probing protein/protein interactions which makes use of the bacterial flagellum to display random peptide libraries on the surface of E. coli. In developing the system the entire coding sequence of E. coli thioredoxin (trxA) was inserted into a dispensable region of the gene for flagellin (fliC), the major structural component of the E. coli flagellum. The resulting fusion protein (FLITRX) was efficiently exported and assembled into partially functional flagella on the bacterial cell surface. A diverse library of random dodecapeptides were displayed in FLITRX on the exterior of E. coli as conformationally constrained insertions into the thioredoxin active-site loop, a location known to be a highly permissive site for the insertion of exogenous peptide sequences into native thioredoxin. To demonstrate that members of this library could be bound and selected via specific protein/protein interactions to a target protein, a method was devised to enable efficient isolation of those bacteria displaying peptides with affinity to immobilized antibodies. We have unambiguously mapped three different antibody epitopes using this method. Peptides selected as FLITRX active-site fusions retain their binding specificity when made as native thioredoxin active-site loop fusions. This will facilitate future structural characterizations and broaden the general utility of the system for exploring other classes of protein-protein interactions.

Amino Acid Sequence↗
PubMed1995

Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA.

Enterokinase (EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum. The enzyme acts by converting trypsinogen to trypsin via a highly specific cleavage following the pentapeptide recognition sequence (Asp)4-Lys. This stringent site specificity gives EK great potential as a fusion protein cleavage reagent. Recently, a cDNA encoding the catalytic (light) chain of bovine enterokinase (EKL) was identified, characterized, and transiently expressed in mammalian COS cells. We report here the production of EKL in Escherichia coli by a novel secretory expression system that utilizes E. coli DsbA protein as an N-terminal fusion partner. The EKL cDNA was fused in-frame to the 3'-end of the coding sequence for DsbA, with the two domains of the fusion protein separated by a linker sequence encoding an enterokinase recognition site. Active, processed recombinant EKL (rEKL) was generated from this fusion protein via an autocatalytic cleavage reaction. The enzymatic properties of the bacterially produced rEKL were indistinguishable from the previously described COS-derived enzyme. Both forms of rEKL were capable of cleaving peptides, polypeptides and trypsinogen with the same specificity exhibited by the native heterodimeric enzyme purified from bovine duodena. Interestingly, rEKL activated trypsinogen poorly relative to the native heterodimeric enzyme, but was superior in its ability to cleave artificial fusion proteins containing the (Asp)4-Lys recognition sequence.

Amino Acid Sequence↗
PubMed1993

Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase.

Enterokinase (enteropeptidase) is a heterodimeric serine protease that is responsible for the physiological activation of trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys. In this paper, we report the cloning and functional expression of a cDNA encoding the catalytic domain (light chain) of bovine enterokinase. The nucleotide sequence of this cloned cDNA predicts a 235-amino acid polypeptide that shares a high degree of homology with a variety of mammalian serine proteases involved in digestion, coagulation, and fibrinolysis. We have developed a novel expression method for the enzyme which utilizes the secretory leader and propeptide of the mammalian serine protease PACE fused to the enterokinase light chain amino terminus. Efficient cleavage of the paired dibasic amino acid cleaving enzyme (PACE) propeptide was achieved by coexpression with human PACE or yeast KEX2. The mature product migrates at 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comparable to light chain derived from bovine duodena, and exhibited high levels of activity in cleaving the enterokinase-specific fluorogenic substrate Gly-(Asp)4-Lys-beta-naphthylamide. The recombinant single-chain form of enterokinase was also capable of activating trypsinogen, indicating that the specificity of the enzyme for its natural substrate is retained even in the absence of the noncatalytic enterokinase heavy chain.

Amino Acid Sequence↗
PubMed1993

A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm.

We have developed a versatile Escherichia coli expression system based on the use of E. coli thioredoxin (trxA) as a gene fusion partner. The broad utility of the system is illustrated by the production of a variety of mammalian cytokines and growth factors as thioredoxin fusion proteins. Although many of these cytokines previously have been produced in E. coli as insoluble aggregates or "inclusion bodies", we show here that as thioredoxin fusions they can be made in soluble forms that are biologically active. In general we find that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels. Two additional properties of E. coli thioredoxin, its ability to be specifically released from the E. coli cytoplasm by osmotic shock or freeze/thaw treatments and its intrinsic thermal stability, are retained by some fusions and provide convenient purification steps. We also find that the active-site loop of E. coli thioredoxin can be used as a general site for small peptide insertions, allowing for the high level production of soluble peptides in the E. coli cytoplasm.

Amino Acid Sequence↗
PubMed1991

The structure of expert diagnostic knowledge in occupational medicine.

Development of an artificial intelligence expert system for diagnosing occupational lung disease requires explicit specification of the structure of knowledge necessary in clinical occupational medicine independent of the process by which the knowledge is utilized. Furthermore, explicit recognition of sources of uncertainty is necessary. Seven categories of knowledge define the diagnostic knowledge base in occupational pulmonary medicine. These include four objects (jobs, industries, exposures, and diseases) and three relationships between pairs of objects. This analysis demonstrates some of the unique aspects of occupational medicine expertise.

Algorithms↗
PubMed1991

Status report on ambulatory care and education in the VA Western Region and western medical schools.

Since the initial 1988 Ambulatory Care and Education (ACE) conference, reported in the October 1989 supplement to Academic Medicine, the Western Region Veterans Health Administration and its 11 affiliated western medical schools have established several programs and related activities that implement strategic ACE recommendations. This report gives an update on the ACE Advisory Group; the Pilot Ambulatory Care and Education (PACE) Center at the VA Medical Center, Sepulveda, California; other innovative ambulatory care and education projects; the second ACE Development Conference; and future activities in the expanded Western Region of the Department of Veterans Affairs.

Ambulatory Care↗
PubMed1991

Artificial intelligence-assisted occupational lung disease diagnosis.

An artificial intelligence expert-based system for facilitating the clinical recognition of occupational and environmental factors in lung disease has been developed in a pilot fashion. It utilizes a knowledge representation scheme to capture relevant clinical knowledge into structures about specific objects (jobs, diseases, etc) and pairwise relations between objects. Quantifiers describe both the closeness of association and risk, as well as the degree of belief in the validity of a fact. An independent inference engine utilizes the knowledge, combining likelihoods and uncertainties to achieve estimates of likelihood factors for specific paths from work to illness. The system creates a series of "paths," linking work activities to disease outcomes. One path links a single period of work to a single possible disease outcome. In a preliminary trial, the number of "paths" from job to possible disease averaged 18 per subject in a general population and averaged 25 per subject in an asthmatic population. Artificial intelligence methods hold promise in the future to facilitate diagnosis in pulmonary and occupational medicine.

Artificial Intelligence↗
PubMed1989

Quality of care in family practice: does residency training make a difference?

As the proportion of physicians who enter residency training in family practice steadily increases, so does the need to evaluate the impact of their training and postgraduate education on the quality of care in their practices. We audited the practices of 120 randomly selected family physicians in Ontario, who were separated into four groups: nonmembers of the College of Family Physicians of Canada (CFPC), members of the CFPC with no certification in family medicine, certificated members without residency training in family medicine and certificated members with residency training in family medicine. The practices were assessed according to predetermined criteria for charting, procedures in periodic health examination, quality of medical care and use of indicator drugs. Generally the scores were significantly higher for CFPC members with residency training in family medicine than for those in the other groups, nonmembers having the lowest scores. Patient questionnaires indicated no difference in satisfaction with specific aspects of care between the four groups. Self-selection into residency training and CFPC membership may account for some of the results; nevertheless, the findings support the contention that residency training in family medicine should be mandatory for family physicians.

Adult↗
PubMed1989

Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching.

We have analyzed the kinetics of DNA synthesis catalyzed by reverse transcriptase from human immunodeficiency virus 1 (HIV-1). Reverse transcriptase, overproduced in Escherichia coli and purified to homogeneity, has polymerase and RNase H activity. Reverse transcriptase forms a stable complex with poly(rA).oligo(dT) primer-templates in the absence of Mg2+ and dTTP with an equilibrium dissociation constant of 3 nM. Synthesis from these preformed complexes can be initiated, and restricted to a single processive cycle, by the simultaneous addition of Mg2+, dTTP, and excess competitor RNA. Preformed complexes decay with a maximal half-life of 2-3 min. Synthesis on poly(rA) templates is processive with an incorporation rate of 10-15 nucleotides/s at 37 degrees C. Processivity varies widely with the template used, increasing from a few to greater than 300 nucleotides in the order: poly(dA) less than double-stranded DNA less than single-stranded DNA less than single-stranded RNA less than poly(rA). On double-stranded DNA reverse transcriptase catalyzes limited strand-displacement synthesis of up to 50 nucleotides. On RNA-DNA hybrids significant DNA synthesis is observed only after degradation of the RNA strand by the RNase H activity of reverse transcriptase. Intermolecular strand switching occurs with poly(rA) templates. At low ionic strength reverse transcriptase can use multiple templates with a single primer, leading to products of greater than template length. Reverse transcriptase and primer do not have to dissociate during the exchange of template strands, thus allowing processive DNA synthesis across template borders.

DNA, Single-Stranded↗
PubMed1989

Evaluating preclinical medical students by using computer-based problem-solving examinations.

A study was designed to determine the feasibility of creating and administering computer-based problem-solving examinations for evaluating second-year medical students in immunology and to determine how students would perform on these tests relative to their performances on concurrently administered objective and essay examinations. The results indicate that problem-solving questions of uniform difficulty can be created and that correct solution of the problems correlates with the early selection of the most relevant data. The mean scores on the three types of tests were not significantly different. The scores on the essay and objective examinations were most highly correlated, those on the computer-based and objective examinations were minimally correlated, and those on the computer-based and essay examinations were not correlated.

Allergy and Immunology↗
PubMed1989

Predicate calculus, artificial intelligence, and workers' compensation.

Application of principles of predicate calculus (PC) and artificial intelligence (AI) search methods to occupational medicine can meet several goals. First, they can improve understanding of the diagnostic process and recognition of the sources of uncertainty in knowledge and in case specific information. Second, PC provides a rational means of resolving differences in conclusion based upon the same premises. Third, understanding of these principles allows separation of knowledge (facts) from the process by which they are used and therefore facilitates development of AI-based expert systems. Application of PC to recognizing causation of pulmonary fibrosis is demonstrated in this paper, providing a method that can be generalized to other problems in occupational medicine. Application of PC and understanding of AI search routines may be particularly applicable to workers' compensation where explicit statement of rational and inferential process is necessary. This approach is useful in the diagnosis of occupational lung disease and may be particularly valuable in workers' compensation considerations, wherein explicit statement of rationale is needed.

Artificial Intelligence↗
PubMed1988

Ameloblastoma.

Explore the source record for details and available documents.

Adult↗
PubMed1987

Structure-function studies on bacteriorhodopsin. I. Expression of the bacterio-opsin gene in Escherichia coli.

To express the bacterio-opsin (bop) gene in Escherichia coli, we have employed the inducible expression vectors pIN-II-A, -B, and -C (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The vectors contain three cloning sites early in the E. coli lipoprotein gene (lpp) which is transcribed from tandem lpp and lac promoters. The bop gene was modified so as to delete the N-terminal leader sequence and then cloned into each of the three cloning sites to encode three different lipoprotein/bacterio-opsin fusions. Expression of the fusions was demonstrated both in vitro and in vivo. The fusion protein was estimated to be about 0.05% of the total cell protein. The cause for the low level of expression apparently was neither an inadequate level of mRNA nor degradation of the protein. However, expression of the fusions caused inhibition of the growth of the host to varying extents. One fusion protein was purified from E. coli membranes to homogeneity by immunoaffinity chromatography followed by preparative gel electrophoresis. The purified fusion protein generated a bacteriorhodopsin-like chromophore on treatment with defined lipid/detergent mixtures and retinal. When reconstituted into vesicles, the protein pumped protons on illumination comparably to the reconstituted native bacterio-opsin.

Bacteriorhodopsins↗