Autolysin

Saul Purton S.Purton at ucl.ac.uk
Fri Jul 7 07:01:17 EST 1995


Dear All,

Recently I asks for tips on making autolysin. Thanks for all the replies,
some of which are given below. Clearly, the critical thing is very high %
mating.

___________________________________

From: Karen Kindle <karen_kindle at qmrelay.mail.cornell.edu>

I think I have the opposite problem with autolysin-too much activity which can
be a problem.  We need to dilute our autolysin for many applications.  I use
high efficiency mating cells (CC620 and CC621) and grow them phototrophically,
as described in the Sourcebook.  They are then starved for nitrogen overnight
and then the two mating types are mixed together for 1-2 hours.  I pretty much
follow the instructions, except I simply do a high speed (20K) spin, then
filter through 0.45 micron filter, then freeze.  Very simple except for
manipulating the large amounts of culture supernatant.
___________________________________

From:Bill Snell <WSNELL at MEDNET.SWMED.EDU>

Making g-lysin preps is not always easy.  We have published a method in
Experimental Cell Research (Buchanan and Snell, "Biochemical studies
on lysin, a cell wall degrading enzyme released during fertilization in
Chlamydomonas; ECR 179:181-193) that works almost all of the time.  We
understand why most details of the method are important and we can
only guess about others.  For example you need to have highly
competent gametes (greater than 70-90% of them can fuse), we're not
sure why using cells at 108 is so important, but it is.  Also we're not sure
about why the 0.5 hr mixing time is important, but it may have to do with
the fact that g-lysin may bind to cell walls.  If the cells are mixed long
enough in the presence of lysin, the walls become degraded and are not
sedimented when you clarify the lysin.
     Anyway, most times we can change some of these parameters and
get good lysin.  But, if our lysin preps start to be less efficient, we go
back to being fastidious and it works.

___________________________________

From: kosuke at andrew.stanford.edu (Kosuke Shimogawara)

By my experience, yield of the lytic activity is completely dependent
with the efficiency of mating.  To get good mating, I am using good
mating strains, i.e., CC620 and CC621, both of them are available form
the Chlamydomonas Genetic Center.  You might also be very careful for the
concentration of the cells during mating.  If the concentration during
mating is too high, the efficiency of mating seems to become worse
(probably anaerobic environment is harmful).

The following is my protocol.

- Cultivate both mating type of the cells up to about 3x10^6 /mL in 250
mL of TAP in 1 L flask.
- Collect the cells by centrifugation at 3000 rpm 5 min.
- Resuspend the cells in 1L of TAP-N (NH4Cl was replaced with the same
concentration of KCl) in 2 L flask.
- Induce gamete by gently shaking for 24 hr under light.
- Harvest the cells by centrifugation at 3000 rpm 5 min.
- Resuspend each cells in 200 mL of TAP-N.
- Mix both mating type of cells in 2 L flask (larger vessel seems
better).
- Keep it under light without shaking for 1 to 2 hr to allow mating
(vigorous shaking seems harmful).
- Remove the cells by centrifugation at 8000 rpm.
- Remove cell debris by centrifugation at max speed.
- Filtrate the medium by 0.45 um membrane filter unit.
- Keep in -80C until use.

___________________________________

Saul Purton
Dept. of Biology
University College London
Gower Street
London WC1E 6BT, U.K.

E-mail: S.Purton at ucl.ac.uk
Phone:  0171-387 7050 x 2675
FAX:    0171-380 7096






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