I am purifying a native bacteriocin protein from an induced E. coli lysate.
After following a protocol from a paper for almost 6 months, my lack of success in purification was fixed by actually contacting the author. However, I learned that the Q sepharose AIEX column in the paper was chosen because it was applicable to many other projects, and not optimized for the protein I am trying to purify.
I am using a Bio-Rad NGC Quest (with additional column switching module)
My protein has a pI of 9.69, 42kDa.
I am using preliminary industrial methods for purification, using 2L culture, inducing, centrifuging, and collecting the supernatant containing my protein. This was then concentrated and buffer exchanged into 10mM Tris-Cl pH 8 by tangential flow 30kDa cutoff cassette. The paper used 10mM Tris-Cl buffer pH 8 and +1M NaCl Elution buffer to equilibrate a Q column. According to this information, the protein's exposure to a lower buffer pH makes it positively charged and able to bind to a CIEX column. However, the protein did actually bind to the Q column (along with many contaminants) but was active in a few fractions determined by drop test activity assay. SDS-PAGE showed my protein (in a low amount) with a great deal of bands in the eluent.
Realizing that the paper was wrong, I buffer exchanged the sample (by ultrafiltration) I had (which was in 10mM Tris-Cl pH 8) into 25mM sodium phosphate pH 8 and prepared the same buffers (25mM sodium phosphate pH 8) for use on a CIEX 1mL HiTrap SP XL, but everything came out in the flowthrough. Did this not work because my initial buffer was not sodium phosphate? What buffer should I use for my E coli protein?
I suspect that the protein is binding to another one so I am thinking about supplementing the buffer with EDTA, DTT, TX-100, or NaCl to keep them separated. Would the supplemented buffer be okay to run on the FPLC? Keep in mind my induction method does not require me to lyse any cells**
I am hoping to find a proper buffer to use before just supplementing the one I use currently because I do not want to waste 2L cultures just trying different buffers. Hopefully someone here can help!