ORIGINAL PAPER M. C. Loewen AE X. Liu AE P. L. Davies AE A. J. Daugulis Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris Received: 19 November 1996 / Received revision: 29 January 1997 /Accepted: 7 March 1997 Abstract Sea raven type II antifreeze protein (SRAFP) is one of three di��erent 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-dimen- sional structure of SRAFP, study its ice-binding mech- anism, and as a basis for engineering these molecules, an e��cient system for its biosynthetic production was de- veloped. Several di��erent 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 di��erent strate- gies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Muts clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut+ clones. The mixed- feeding/Muts strategy proved to be the most e��cient with SRAFP yields reaching 30 mg/l. Introduction Antifreeze proteins (AFP) have been isolated from a number of organisms including fish, insects, plants and bacteria (Davies and Hew 1990 Sun et al. 1995 Gri��th et al. 1992 Tomchaney et al. 1982). These molecules protect the freeze-intolerant organisms from freezing in sub-zero conditions by adsorption of the protein to the surface of seed ice-crystals resulting in inhibition of ice- crystal growth (Raymond and DeVries 1977). The via- bility of freeze-tolerant organisms is helped by the ability of AFP to inhibit ice recrystallization (Carpenter et al. 1992). Type II AFP, one of three di��erent fish AFP identified to date (Davies and Hew 1990), has been isolated from three distantly related fish, including sea raven, herring and smelt. The supply of this AFP from natural sources is scarce, severely limiting biotechno- logical applications and research into the structure and mechanism of action. Accordingly it is essential to de- velop a reliable source of defined recombinant type II AFP. A number of di��erent expression systems have been tested for expression of sea raven type II antifreeze protein (SRAFP). A recombinant baculovirus-vector- infected army worm cell system was initially chosen for its ability to secrete and correctly fold disulfide-bonded proteins (O���Reilly et al. 1992). The T7-promoter-driven expression system in Escherichia coli was also tested on the basis of the previous success achieved in expressing mannose-binding protein, a lectin domain homolog to SRAFP (Ewart et al. 1992), in E. coli (Weis et al. 1991). Finally the Pichia pastoris yeast expression system was tested on the basis of its ability to produce very high yields of foreign proteins (Barr et al. 1992 Digan et al. 1989 Clare et al. 1991 Cregg et al. 1993). In the Pichia system (Invitrogen, San Diego, Calif.) the gene for expression is placed immediately down- stream of the strong yeast alcohol oxidase promoter, which is repressed by glycerol and glucose and induced by methanol. The Pichia system allows for the genomic integration of foreign genes, thus precluding di��culties associated with plasmid-based expression, such as plas- mid instability. In addition, integration of the expression cassette can be directed to two alternative sites in the Appl Microbiol Biotechnol (1997) 48: 480���486 �� Springer-Verlag 1997 This paper is dedicated to the memory of Xiaoting Liu M. C. Loewen AE P. L. Davies Department of Biochemistry, Queen���s University, Kingston, Ontario, Canada, K7L 3N6 X. Liu AE A. J. Daugulis (&) Department of Chemical Engineering, Queens University, Kingston, Ontario, Canada, K7L 3N6 Tel.: (613)545 2784 Fax: (613)545 6637 e-mail: Daugulis@chee.queensu.ca

yeast genome. In one case the methylotrophic phenotype of the yeast is maintained (providing a Mut+ pheno- type), and in the other the methylotrophic phenotype is deleted (Muts phenotype). Thus methanol can be used as a carbon source and/or inducer of foreign gene expres- sion. This ability provides a readily controllable system for expression of foreign proteins. In this work we describe the shake-flask expression of the cystine-rich SRAFP from Pichia, and demonstrate that the recombinant protein has identical activity to SRAFP isolated from sea raven serum. This is compared to expression levels obtained from E. coli and baculo- virus systems. We further describe the development of expression from yeast by fermentation techniques. Through several fermentation strategies, we have de- veloped an e��cient system for the biosynthetic expres- sion of wild-type and mutant SRAFP in quantities su��cient for testing biotechnological applications, X-ray crystallography and isotopic labelling for nuclear mag- netic resonance (NMR) studies. Materials and methods Bacterial and yeast strains The methylotrophic strain of P. pastoris, GS115(his4) commercially availablefromInvitrogen(SanDiego,Calif.,US)(Creggetal.1985), was used for expression of recombinant type II AFP. E. coli JM83 was used for all plasmid constructions and propagations. Media compositions Bu��ered minimal glycerol complex medium (BMGY) contained the following (per liter): yeast extract (10 g), meat peptone (20 g), 100 mM potassium phosphate bu��er pH 6.0, yeast nitrogen base without amino acids (13.4 g), biotin (400 lg), glycerol (10 ml). Bu��ered minimal methanol complex medium (BMMY) contained the same components as BMGY except that glycerol was replaced by methanol (5 ml/l). Minimal methanol medium (MM) contained the following (per liter): yeast nitrogen base without amino acids (13.4 g), biotin (400 lg), methanol (5 ml). Minimal glycerol medi- um (MG) was the same as MM medium except that methanol was replaced by glycerol (10 ml/l). Fermentation medium contained the following (per liter): glycerol (50 g), ammonium sulfate (20 g), KH2PO4 (12 g), MgSO4 AE 7H2O (4.7 g), CaCl2 AE 2H2O (0.36 g), plus trace elements as follows: CaSO4 AE 5H2O 0.2 lM, KI 1.25 lM, MnSO4 AE 4H2O 4.5 lM, Na2MoO4 AE 2H2O 2.0 lM, H3BO3 0.75 lM, ZnSO4 AE 7H2O 17.5 lM, FeCl3 AE 6H2O 44.5 lM. The pH was adjusted to 5.5 using 5 M KOH. Plasmid construction The vector pPIC9, which contains the his4 gene for selection in the HIS4) strain, GS115(his4) and the ampicillin-resistance gene for selection in E. coli (Cregg et al. 1985), was used to express the mature sea raven cDNA in P. pastoris. The 5��-end primer, SR1 (5��- GAATTCGAATTCCAGAGAGCCCCACCA-3��), which intro- duced tandem EcoRI sites immediately upstream of the Gln codon at the N terminus of mature SRAFP (Hayes et al. 1989), and the 3�� primer, SR2 (5��-ATACGTAAGCGGCCGCCAAACACCCACT- AAAGTG-3��), which introduced a NotI site 47 base pairs down- stream of the stop codon, were used to amplify the mature AFP cDNA by the polymerase chain reaction (PCR) from the clone pT7-7F-SRm. pT7-7F-SRm is a derivative of the E. coli expression vector pT7-7 (Tabor 1990), in which the mature cDNA of type II AFP was cloned into the NdeI/HindIII sites of pT7-7F. The pT7- 7F vector contains an oriF site for production of single-strand DNA as previously described (DeLuca and Elce 1993). A 492-base- pair product was obtained from PCR and was cloned into the EcoRI and NotI sites of the pPIC9 vector, 24 bases (eight amino acids) downstream from the cleavage site of the a mating factor signal peptide, to give pPIC9-SRm. In addition, the vector pT7-7F- SRm was mutated using a primer-directed mutagenic method (Kunkel et al. 1987) with primer CTHT1 (P5��-TGCGCCATG- ACATTCCACCACCACCACCACCACTAAGCGGCCGCTGA- GCTAACACAGAGG-3��), which introduced a six-histidine-long His tag at the C-terminal end of the cDNA, immediately prior to the stop codon to allow simplified protein purification by a��nity chromatography to a nickel column. The primer also adds a NotI site immediately following the stop codon. A fragment, that in- cluded the His tag was excised using the NotI site and a unique BstEII site further upstream within the cDNA. This was cloned into the corresponding sites within the pPIC9-SRm vector to pro- duce the final expression construct, pPIC9-SRm-CTHT, which di��ered from pPIC9-SRm by the presence of the C-terminally coded His-tag. Transformation and induction Vector DNA (10 lg), produced in E. coli, was linearized and transformed into GS115(his4). Vector linearized by BglII was in- tegrated by homologous recombination into the AOX1 gene site producing clones with a His+, methanol-utilization-slow (Muts) phenotype. Vector linearized at the SalI site integrated into the his4 gene site of the GS115(his4) genome, without disruption of the his4 plasmid gene, gave a His+, Mut+ phenotype. The linearized vector was introduced into P. pastoris using the spheroplast transforma- tion procedure essentially as described by Cregg et al. 1985. Transformant cultures (50 ml) were grown in non-ba��ed shake flasks (250-ml conical flasks) at 250 rpm and 30 ��C in a standard New Brunswick Scientific environmental incubator shaker (Edison, N.J.) for 48 h in BMGY medium, which contained glycerol and therefore inhibited the alcohol oxidase promoter. Cells were har- vested by centrifugation and resuspended in the same volume of BMMY, which contained methanol for induction of the alcohol oxidase promoter, and again incubated for 48 h at 30 ��C with shaking. Cells were removed by centrifugation, leaving recombi- nant AFP to be purified from the medium. Analysis of AFP AFP samples were analyzed by electrophoresis on a 15% poly- acrylamide/sodium dodecyl sulfate gel, containing 0.1 M sodium phosphate and 4 M urea, at pH 6.8, and blotted onto a nylon membrane (PVDF, NEN). The membrane was incubated with rabbit anti-(sea raven AFP) antiserum and then horseradish-per- oxidase-linked goat anti-(rabbit IgG). Detection was carried out using enhanced chemiluminescence. Quantification was carried out by visual inspection of band intensities compared to intensities of standards. Measurements of the antifreeze activity were made in 100 mM NH4HCO3 (pH 7.9) using a nanoliter osmometer (Clifton Tech- nical Physics, Hartford, New York) as described by Chakrabartty and Hew (1991). Antifreeze activity is defined as thermal hysteresis, which is the di��erence (��C) in temperatures at which the seed crystal grows and shrinks. Ice crystal growth and morphology were observed using a Panasonic closed-circuit TV camera attached to a Leitz dialux 22 microscope and recorded by a JVC Super VHS video recorder. Still photographs of ice crystals were taken 1 min after the formation of the bipyramidal ice crystal under 0.1 ��C supercooling. Fermentation Inoculum cultures (600 ml) were grown in MG medium for 48 h at 30 ��C, in 2-l conical flasks, at 250 rpm. This 10% inoculum was 481