ABRF-ESRG'03: analysis of a PVDF-bound known protein with a homogeneous amino-terminus.

The Association of Biomolecular Resource Facilities 2003 Edman Sequencing Research Group (ABRF-ESRG'03) sample is the 15th in a series of studies designed to allow participating members to evaluate their abilities to analyze the N-terminus of a protein or peptide using automated Edman degradation chemistry. It is a follow-up study to the ESRG'02 sample, which was a single protein with a heterogeneous N-terminus. Both the 2002 and 2003 samples were obtained from the same protein complex and were resolved by SDS-PAGE followed by electrophoretic transfer to PVDF membrane. The ABRF-ESRG'03 sample had an apparent molecular weight of 49 kDa and a single N-terminus, with initial yields of approximately 2 pmol. Participants were asked to sequence 25 residues and return their results to the ESRG for analysis along with two completed surveys and an area/pmol table for repetitive and initial yield calculations. Data for 46 responses are presented which include initial yields, repetitive yields, sequencer performance, and ability to identify the protein.

American oyster, Crassostrea virginica, expresses a potent antibacterial histone H2B protein.

An antibacterial protein was purified from acidified gill extract of a bivalve mollusk, the American oyster (Crassostrea virginica). Protein isolation was best accomplished by briefly boiling the tissues in a weak acetic acid solution. Adding protease inhibitors while boiling did not have a major effect on activity recovery. In contrast, use of only protease inhibitors (without boiling) resulted in virtually no recovery of this activity. The amino acid sequence of this antibacterial protein was identified as a histone H2B and was designated cvH2B. cvH2B had potent activity against gram-negative bacteria, including the human pathogens Vibrio parahaemolyticus and Vibrio vulnificus, which commonly reside in oyster tissues. We estimated that the concentration of this protein was well within the concentration that was inhibitory to these bacterial pathogens in vitro. This is the first report of the antimicrobial function of histone H2B from any mollusk.

Purification of a novel arthropod defensin from the American oyster, Crassostrea virginica.

An antimicrobial peptide was purified from acidified gill extract of a bivalve mollusk, the American oyster (Crassostrea virginica), by preparative acid-urea--polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography. The 4265.0 Da peptide had 38 amino acids, including 6 cysteines. It showed strongest activity against Gram-positive bacteria (Lactococcus lactis subsp. lactis and Staphylococcus aureus; minimum effective concentrations [MECs] 2.4 and 3.0 microg/ml, respectively) but also had significant activity against Gram-negative bacteria (Escherichia coli D31 and Vibrio parahemolyticus; MECs 7.6 and 15.0 microg/ml, respectively). Comparison of the amino acid sequence with those of other known antimicrobial peptides revealed that the novel peptide had high sequence homology to arthropod defensins, including those from other bivalves, the mussels Mytilus edulis and Mytilus galloprovincialis. This is the first antimicrobial peptide to be isolated from any oyster species and we have named it American oyster defensin (AOD).

ABRF-2002ESRG, a difficult sequence: Analysis of a PVDF-bound known protein with a heterogeneous amino-terminus.

The ABRF-2002 Edman Sequencing Research Group (ESRG) sample (ABRF-2002ESRG) was the 14th in a yearly series designed as an education and self-evaluation tool for laboratories performing Edman sequence analysis. This year's study used a known protein with a heterogeneous amino-terminus, and thus was one of the more challenging protein samples distributed by an Association for Biomolecular Resource Facilities (ABRF) research group. The sample was originally submitted to an ESRG member's lab, and after analysis was thought to demonstrate an analytical problem that would be of interest to the general sequencing community. The protein was purified using commercially available, pre-cast sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride.Protein bands were distributed to 72 members of the ABRF who requested ABRF-2002ESRG, along with a data instruction sheet and a brief survey. Participating members were requested to report observed raw data, interpret the data as they normally would for an investigator, and identify the protein using a database search. Study results from 31 responses are presented to show how labs fared with a difficult but sequenceable sample.