Anti-Microbial Protein
Added: Mar 28, 2012 8:30 am The USDA, Agricultural Research Service (ARS), Animal Biosciences and Biotechnology Laboratory (ABBL) has a requirement for a company to provide it with 12 grams of >98% pure, endotoxin-free, Lysotaphin-TAT-His6 (Lyso-TAT), a fusion of lysostaphin to the HIV TransActivator of Transcription (TAT) protein transduction domain with a C-terminal 6 x His tag for nickel column purification. The fusion protein (~270 residues) is encoded on the pET21a (Novagen) IPTG inducible plasmid. The ARS has a protocol using NiNTA nickel column matrix (Qiagen) that yields ~2 mg Lyso-TAT per liter from shaker flask grown E. coli harboring the Lyso-TAT vector.This procurement is reserved exclusively for small businesses with a size standard of no more than 1,000 employees, referencing (NAICS) North American Industrial Classification Code 325199. Prior to awarding the contract, the project must include a demonstrated purification on a small volume (e.g. 10 liter) of fermentation grown E.coli BL21 (DE3) to demonstrate that the target amount (12 g) can be produced at the concentration and quality anticipated for the full scale grow up. Documentation of the company's capability must be submitted by April 18, 2012. Upon verification of the company's submission, a contract will be awarded and the final product of the required 12 grams of the material is required by June 8, 2012. The USDA will send the Lyso-TAT encoding plasmid and purification protocol to interested parties upon their written request. Requests for the encoding plasmid and protocol must be submitted to Dr. David Donovan via email at david.donovan@ars.usda.gov. The final product must be demonstrated via SDS PAGE and OD280 to be >98% pure, at a concentration of >10 mg/ml Lyso-TAT, endotoxin free, in: 50mM NaH2PO4300mM NaCl250mM imidazole30% glycerolpH 8.0 The ARS will not pay for the demonstration that the Lyso-TAT protein can be produced in the quantity and quality needed. Such a demonstration will be required prior to awarding the contract. The ARS will provide the Lyso-TAT expression vector, and the company must provide this documentation within tee (3) weeks of receiving the vector, but not later than April 18, 2012. Specifically, the demonstration must provide the following: a. SDS PAGE (15% gel) with the final product and size markers. The loading of the gel should include 4 lanes with 1, 5, 10 and 20 micrograms Lyso-TAT each. The product must be at least 98% pure, at a concentration of >10 mg/ml Lyso-TAT. b. The detection of endotoxin must be performed with the Lonza amoebocyte lysate endotoxin detection kit, http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_ManualsProductInstructions_QCL-1000_Product_Insert.pdf and the final protein solution must achieve < 5 IU endotoxin/ml in the final product.c. Concentration can be determined via any conventional protein detection assay or A280 nm.d. Protein will be nickel column purified and the final product should be in this buffer:50mM NaH2PO4300mM NaCl250mM imidazole30% glycerolpH 8.0e. An aliquot equal to 100 µl of >10 mg/ml will be provided to USDA for in-house verification of the product, prior to the award. The isolation procedure is virtually identical to published protocols in numerous scientific journals from the ARS lab, with minor modifications. (Donovan DM, Foster-Frey J. LambdaSa2 prophage endolysin requires Cpl-7-binding domains and amidase-5 domain for antimicrobial lysis of streptococci. FEMS Microbiol Lett. 2008 Oct;287(1):22-33. ) Responses to this notice must be submitted via email no later than 3 P.M. (EDT) on April 18, 2012, to john.wilkinson@ars.usda.gov and be on company letter head and include: (1) the documentation stated above, along with a substantive statement regarding the company's capability, (2) a list of at least tee (3) references (with contact information) for whom this type of material or related services/material has been previously provided, and (3) cost to provide the 12 grams of material. The sample must also be submitted no later than 3 P.M (EDT) on April 18, 2012 only to Dr. David Donovan at USDA, ARS, ABBL; 10300 BALTIMORE AVENUE; BLDG 230, BARC-EAST, Room 104; Beltsville, MD 20705. The anticipated award date is May 18, 2012. 572099A Department of Agriculture Agricultural Research Service Central Program Planning Coordination and Support
3/28/12, 8:30 AM