Biomedical subjects

Carl A Batt

Publications and source records attributed to Carl A Batt.

21 records · Page 2Linked to original sources

PubMedDate not supplied

Enzymatic surface-initiated polymerization: a novel approach for the in situ solid-phase synthesis of biocompatible polymer poly(3-hydroxybutyrate).

A novel system for surface-initiated enzymatic polymerization of a film of polyhydroxyalkanoate (PHA) on solid surfaces has been developed and characterized. PHAs are aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy, and their properties range from elastomers to thermoplastics, depending on their monomeric composition. The PHA synthase from Ralstonia eutropha H16 was expressed as a poly-histidine fusion in Escherichia coli and immobilized onto several solid substrates through a transition-metal complex, Ni(2+)-nitrilotriacetic acid. The immobilized PHA synthase catalyzed the surface-initiated polymerization of 3-(R)-hydroxybutyryl-CoA, forming a polymer film with a uniform thickness on the surface. In this work, we describe the patterned immobilization of the intact enzyme on silicon and subsequent enzymatic polymerization. The immobilized enzyme had a lower specific activity and did not exhibit a lag phase as compared to the soluble enzyme.

Acyltransferases↗

PubMedDate not supplied

Glycosylation profiles of the human colorectal cancer A33 antigen naturally expressed in the human colorectal cancer cell line SW1222 and expressed as recombinant protein in different insect cell lines.

The A33 antigen is a cell surface glycoprotein expressed in human gastrointestinal epithelium and in 95% of colorectal cancers. We have compared the N-linked glycosylation profile of A33 antigen naturally expressed in a human colorectal cancer cell line with recombinant human A33 antigen (rA33) produced in insect cell culture using the baculovirus expression vector. N-Linked glycans were enzymatically released from the protein, and glycan composition was analyzed by HPLC. In three insect cell lines tested (Sf-21, Tn5B1-4, and Tn-4s), glycosylation of rA33 was dominated by high mannose structures (M5Gn2 to M9Gn2; 78-95% of total N-linked glycans), with M8Gn2 being the single most abundant glycoform. A33 antigen naturally expressed in the SW1222 human colon cancer cell line (A33) also possessed a high abundance of high mannose glycans (72%). No complex glycosylation was detected on rA33 expressed in insect cells. Natural A33 was galactosylated to a small extent (6%). These results illustrate a case of similar glycosylation of a glycoprotein between a recombinant version produced in insect cell culture and its counterpart naturally expressed in human cell culture.

Animals↗

PubMedDate not supplied

Evaluation of Mut+ and MutS Pichia pastoris phenotypes for high level extracellular scFv expression under feedback control of the methanol concentration.

Extracellular secretion of over 4 g x L(-1) of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut+ and MutS phenotypes were compared in a bioreactor under closed-loop PID control of the methanol level. As expected, the MutS phenotype has a growth rate lower than that of the Mut+ (0.37 vs 1.05 d(-1)) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut+ (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g x L(-1) for the MutS also has a positive effect on the final product concentration. Both Mut+ and MutS phenotypes reach a maximum biomass density around 450 g x L(-1) wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOX promoters, without prior knowledge of the culture growth parameters.

Antibodies, Monoclonal↗