http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6V5N-4C8PFBS-2&_us er=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1& _urlVersion=0&_userid=10&md5=aee9aceae8dfa17b363ee2ec634debb0
Osmotic shock is OK, or you can try chlorophorm disruption. You can Google for either to find them. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-476TXJW-CK&_u ser=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1 &_urlVersion=0&_userid=10&md5=836b012efa98a0fe0259708135fc1144 Please note that osmotic shock extraction typically employs EDTA which is obviously bad for IMAC. If you're not worried about also extracting cytoplasmic proteins, then a combination of 2:1 I-PER with B-PER (Pierce) works fine and is compatible with IMAC. Please note that just because you have pelB signal on your protein does not guarrantee that your protein finds its way to the periplasm. Secretion in general and secretion in Gram-negative hosts in particular can be very tricky and sometimes impossible. If you have time/effort available I would heartily recommend Bacillus megaterium as an alternative host - it has good secretory capacity and some of its strains have been selected not to secrete appreciable quantities of proteases which (as people who work with B. subtilis will tell you) can be a right pain in the tuches. There's even a commercially available B. megaterium system that does not require chromosomal integration (again, unlike B. subtilis). Artem -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Matt Colins Sent: Monday, February 09, 2009 12:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protocol for harvesting proteins from bacterial periplasmic space Dear all, I plan to prepare a protein fused to pelB signal peptide and secreted to bacterial periplasmic space. Does anyone have a detailed protocol for harvesting the protein from bacterial periplasmic space? The protein needs to be in the native state and will be purified on a nickel column. I would appreciate your help very much! Matt
