X-Message-Number: 9678
From: Eugene Leitl <>
Date: Sun, 10 May 1998 22:49:46 +0400 (MSD)
Subject: AFP synthesis

http://pout.cwru.edu:8080/~frank/rec_pub.html

Appl Microbiol Biotechnol 1997 Oct;48(4):480-486 

Biosynthetic production of type II fish
antifreeze protein: fermentation by Pichia
pastoris.

Loewen MC, Liu X, Davies PL, Daugulis AJ

Sea raven type II antifreeze protein (SRAFP) is one of three
different fish antifreeze proteins isolated to date. These proteins
are known to bind to the surface of ice and inhibit its growth. To
solve the three-dimensional structure of SRAFP, study its
ice-binding mechanism, and as a basis for engineering these
molecules, an efficient system for its biosynthetic production was
developed. Several different expression systems have been tested
including baculovirus, Escherichia coli and yeast. The latter, using
the methylotrophic organism Pichia pastoris as the host, was the
most productive. In shake-flask cultures the levels of SRAFP
secreted from Pichia were up to 5 mg/l. The recombinant protein
has an identical activity to SRAFP from sea raven serum. In order
to increase yields further, four different strategies were tested in
10-l fermentation vessels, including: (1) optimization of pH and
dissolved oxygen, (2) mixed feeding of methanol and glycerol with
Mut(s) clones, (3) supplementation of amino acid building blocks,
and (4) methanol feeding with Mut+ clones. The
mixed-feeding/Mut(s) strategy proved to be the most efficient
with SRAFP yields reaching 30 mg/l. 


J Surg Oncol 1997 Oct;66(2):114-121 

Chemical adjuvant cryosurgery with
antifreeze proteins.

Koushafar H, Pham L, Lee C, Rubinsky B

BACKGROUND AND OBJECTIVES: Imaging monitored
cryosurgery is emerging as an important minimally invasive
surgical technique for treatment of cancer. Although imaging
allows excellent control over the process of freezing itself, recent
studies show that at high subzero temperatures cells survive
freezing. Antifreeze proteins (AFP) are chemical compounds
that modify ice crystals to needle-like shapes that can destroy
cells in cellular suspensions. The goal of this study was to
determine whether these antifreeze proteins can also destroy cells
in frozen tissue and serve as chemical adjuvants to cryosurgery.
METHODS: Livers from six rats were excised, perfused with
solutions of either phosphate-buffered saline (PBS) or PBS with
10 mg/ml AFP-I, and frozen with a special cryosurgery apparatus.
Lobes were frozen with one or two freeze-thaw cycles and the cell
viability was examined with a two stain fluorescent dye test and
histological assessment. RESULTS: A significant percentage of
hepatocytes survive freezing on the margin of a frozen cryolesion.
AFP significantly increase cellular destruction in that region
apparently through formation of intracellular ice.
CONCLUSIONS: This preliminary study demonstrates that
antifreeze proteins may be effective chemical adjuvants to
cryosurgery. 

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