Publications by authors named "Matthew H Parker"

In planta expression of cell wall degrading enzymes is a promising approach for developing optimized biomass feedstocks that enable low-cost cellulosic biofuels production. Transgenic plants could serve as either an enzyme source for the hydrolysis of pretreated biomass or as the primary biomass feedstock in an autohydrolysis process. In this study, two xylanase genes, Bacillus sp. NG-27 bsx and Clostridium stercorarium xynB, were expressed in maize (Zea mays) under the control of two different promoters. Severe phenotypic effects were associated with xylanase accumulation in maize, including stunted plants and sterile grains. Global expression of these xylanases from the rice ubiquitin 3 promoter (rubi3) resulted in enzyme accumulation of approximately 0.01 mg enzyme per gram dry weight, or approximately 0.1% of total soluble protein (TSP). Grain-specific expression of these enzymes from the rice glutelin 4 promoter (GluB-4) resulted in higher-level accumulation of active enzyme, with BSX and XynB accumulating up to 4.0% TSP and 16.4% TSP, respectively, in shriveled grains from selected T0 plants. These results demonstrate the potential utility of the GluB-4 promoter for biotechnological applications. The phenotypic effects of xylanase expression in maize presented here demonstrate the difficulties of hemicellulase expression in an important crop for cellulosic biofuels production. Potential alternate approaches to achieve xylanase accumulation in planta without the accompanying negative phenotypes are discussed.

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5', four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.

Alpha-Fetoprotein (AFP) is a 68 kDa glycoprotein expressed at high levels by the fetal liver and yolk with transcription repressed to very low levels after birth. Transfer of fetal AFP through the placenta into the circulation of the mother is correlated with remission of rheumatoid arthritis, multiple sclerosis, and other autoimmune disorders. AFP is therefore under development as a biopharmaceutical for the treatment of autoimmune diseases. The clinical evaluation of AFP requires the production of hundreds of grams of highly purified and biologically active protein. We have produced goats that express a form of the human AFP transgene under the control of the beta-casein promoter. In this form of rhAFP, the single N-linked glycosylation site was removed by mutagenesis (N233Q). Here, we describe a purification protocol for this recombinant human (rh)AFP from the milk of these transgenic goats. A three-column procedure was developed to produce gram quantities of highly purified rhAFP. Near- and far-UV circular dichroism spectra of human umbilical cord blood AFP and rhAFP were essentially identical, suggesting that the structure is not affected by removal of the glycosylation site. Furthermore, the cell binding and pharmacokinetics of purified rhAFP were similar to human AFP isolated from cord blood. Our results demonstrate that an active form of rhAFP can be produced on industrial scale by expression in transgenic goat milk.