Atherton, CA, United States
Atherton, CA, United States

Time filter

Source Type

Venkataramani S.,Coleman Softlabs Inc. | Venkataramani S.,Boehringer Ingelheim Pharmaceuticals | Truntzer J.,Coleman Softlabs Inc. | Truntzer J.,University of California at Irvine | Coleman D.R.,Coleman Softlabs Inc.
Journal of Pharmacy and Bioallied Sciences | Year: 2013

Aim: The current work is aimed at understanding the effect of pH on the thermal stability of hen egg white lysozyme (HEWL) at high concentration (200 mg/mL). Materials and Methods: Fourier Transform Infrared (FTIR) Spectroscopy with modified hardware and software to overcome some of the traditional challenges like water subtraction, sample evaporation, proper purging etc., are used in this study. Results: HEWL was subjected to thermal stress at pH 3.0-7.0 between 25°C and 95°C and monitored by FTIR spectroscopy. Calculated T m values showed that the enzyme exhibited maximum thermal stability at pH 5.0. Second derivative plots constructed in the amide I region suggested that at pH 5.0 the enzyme possessed higher amount of α-helix and lower amount of aggregates, when compared to other pHs. Conclusions: Considering the fact that HEWL has attractive applications in various industries and being processed under different experimental conditions including high temperatures, our work is able to reveal the reason behind the pH dependent thermal stability of HEWL at high concentration, when subjected to heat denaturation. In future, studies should aim at using various excipients that may help to increase the stability and activity of the enzyme at this high concentration.


Devi V.S.,Coleman Softlabs Inc. | Devi V.S.,Integrated Biotherapeutics, Inc. | Coleman D.R.,Coleman Softlabs Inc. | Truntzer J.,Coleman Softlabs Inc. | Truntzer J.,University of California at Irvine
Protein Journal | Year: 2011

The purpose of this research is to study the thermal unfolding of high concentration bovine Immunoglobulin G (IgG) under 26 different experimental conditions by Fourier Transform Infrared spectroscopy with improved purge conditions and software calculations. When bovine IgG (25-200 mg/mL) was thermally denatured between pH 4.0 and 8.0, it was observed that at 25 mg/mL concentration, the protein exhibited maximum thermal stability at pH 6.0 and 7.0 as evident from the apparent Tm values. Increasing the concentration from 25 to 100 mg/mL at those pH values increased the thermal resistance of the protein by 2-3 C. But, at 200 mg/mL, IgG showed a small decrease in its transition temperature. Presence of 100 mM Trehalose enhanced the Tm values at all conditions and possibly prevented the complete loss of IgG as insoluble aggregates at higher temperatures. Second derivative plots were constructed to explain the conformational changes of IgG during thermal unfolding. © 2011 Springer Science+Business Media, LLC.


PubMed | Coleman Softlabs Inc.
Type: Journal Article | Journal: Current microbiology | Year: 2011

Helicobacter pylori infection increases the risk of cardiovascular diseases besides leading to duodenal and gastric peptic ulcerations. H. pylori cysteine-rich protein B (HcpB) is a disulfide-rich repeat protein that belongs to the family of Sel1-like repeat proteins. HcpB contains four pairs of anti-parallel alpha helices that fold into four repeats with disulfide bonds bridging the helices of each repeat. Recent in vitro oxidative refolding of HcpB identified that the formation and folding of the disulfide bond in the N-terminal repeat are the rate limiting step. Here we attempted to understand the disulfide formation of HcpB in the periplasm of Escherichia coli. The protein was expressed in wild type (possessed enzymes DsbA, B, C, and D) and knock out (Dsb enzymes deleted one at a time) E. coli strains. The soluble part of the periplasm when analyzed by SDS-PAGE and Western Blot showed that the wild type and DsbC/D knock out strains contained native oxidized HcpB while the protein was absent in the DsbA/B knock out strains. Hence the recombinant expression of HcpB in E. coli requires DsbA and DsbB for disulfide bond formation and it is independent of DsbC and DsbD. Prolonged cell growth resulted in the proteolytic degradation of the N-terminal repeat of HcpB. The delayed folding of the N-terminal repeat observed during in vitro oxidative refolding could be the reason for the enhanced susceptibility to proteolytic cleavage in the periplasm. In summary, a good correlation between in vivo and in vitro disulfide bond formation of HcpB is observed.


PubMed | Coleman Softlabs Inc.
Type: Journal Article | Journal: The protein journal | Year: 2011

The purpose of this research is to study the thermal unfolding of high concentration bovine Immunoglobulin G (IgG) under 26 different experimental conditions by Fourier Transform Infrared spectroscopy with improved purge conditions and software calculations. When bovine IgG (25-200mg/mL) was thermally denatured between pH 4.0 and 8.0, it was observed that at 25mg/mL concentration, the protein exhibited maximum thermal stability at pH 6.0 and 7.0 as evident from the apparent T(m) values. Increasing the concentration from 25 to 100mg/mL at those pH values increased the thermal resistance of the protein by 2-3C. But, at 200mg/mL, IgG showed a small decrease in its transition temperature. Presence of 100mM Trehalose enhanced the T(m) values at all conditions and possibly prevented the complete loss of IgG as insoluble aggregates at higher temperatures. Second derivative plots were constructed to explain the conformational changes of IgG during thermal unfolding.

Loading Coleman Softlabs Inc. collaborators
Loading Coleman Softlabs Inc. collaborators