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. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=9678