Progress 08/01/04 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Department of Biological Sciences, University of South Carolina. Additional details of research can be found in the report for the parent project 1275-22000-234-00D Chemical Signals for Managing Insects. An Odorant Binding Protein (OBP), Lygus antennal protein (LAP), was previously identified and cloned. LAP is expressed in the antenna of the hemipteran insect Lygus lineolaris, an important pest on several North American crops, including cotton and beans. LAP cDNA was inserted into the pET22b bacteria expression system as confirmed by DNA sequencing. This DNA construct was transformed into Rosetta Blue (DE3) cells and expression of LAP was activated using IPTG. Previous studies (Dickens et al., 1998) indicated that native LAP protein migrated on SDS gels at an apparent molecular weight (MW) of 15-17 kDa, although the MW was
later determined from sequence analysis to be closer to 12.5 kDa. Bacteria containing LAP-pET22b were first grown at 37C to an OD600 of 0.6, at which time the sample was divided and IPTG added to one sample to a concentration of 1mM. Both samples were then grown over night (~15 hrs) at 27C and the bacteria then harvested. The pET22b vector directs expressed protein to be secreted from the bacteria cytoplasm into the periplasmic space where it is trapped. The bacteria were pelleted (10, 000 x g for 10 min), the pellets dissolved in SDS sample buffer and subjected to SDS polyacrylamide gel electrophoresis (PAGE); proteins were visualized using Coomassie Blue. PAGE showed a prominent induced band running above the 14.5 kDa marker, at an apparent MW that is consistent with that previously reported. We are currently subjecting this protein to immunodetection using LAP antiserum and to purification using his-tag affinity chromatography (the LAP protein is modified by addition of 6
histadines at the C-terminal end for the purpose of manipulating the protein in intended binding assays). This tag is also used to bulk purify the protein from bacterial extracts.
Impacts
(N/A)
Publications
|
Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Department of Biological Sciences, University of South Carolina. Additional details of research can be found in the report for the parent CRIS 1275-22000-234-00D Chemical Signals for Managing Insects. An Odorant Binding Protein (OBP), LAP, was previously identified and cloned. LAP is expressed in the antenna of the hemipteran insect Lygus lineolaris, an important pest on several North American crops, including cotton and beans. We inserted the cDNA encoding the LAP protein into a bacteria expression system (pET22b; Novagen). The protein was expressed in its native form, modified by the addition of a short chain of amino acids (histidines) at the C-terminus. This His-tag allowed rapid purification of the protein. Subsequent affinity studies will use the protein to screen a mixture of environmental odors of possible importance in
tarnished plant bug behavior. These odors will be tested in behavioral and electrophysiological assays aimed at novel attractants and repellents.
Impacts
(N/A)
Publications
|
|