Pichia protocols manual

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Pichia Expression Kit
PROTEIN EXPRESSION A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris Catalog no. K1710-01

Pichia Expression Kit
Version F
160624
25-0043

U.S. Headquarters: Invitrogen Corporation 3985 B Sorrento Valley Blvd. San Diego, CA 92121 Tel: (800) 955-6288 Fax: (619) 597-6201 Internet: tech_service@invitrogen.com

European Headquarters:Invitrogen BV De Schelp 12, 9351 NV Leek The Netherlands Tel: +31 (0) 594 515 175 Fax: +31 (0) 594 515 312 Internet: tech_service@invitrogen.nest.nl

ii

Materials
Kit Contents
Box 1: Spheroplast Module. Store at room temperature. Reagent SOS media Amount 20 ml Components 1 M Sorbitol 0.3X YPD 10 mM CaCl2 Autoclaved, deionized water 1 M Sorbitol 25 mM EDTA, pH 8.0 1 M Sorbitol 10 mM Sodiumcitrate buffer, pH 5.8 1 mM EDTA -1 M Sorbitol 10 mM Tris-HCl, pH 7.5; 10 mM CaCl2 40% (w/v) PEG 3350 (Reagent grade) in water 20 mM Tris-HCl, pH 7.5 20 mM CaCl2

Sterile Water SE SCE

2 x 125 ml 2 x 125 ml 2 x 125 ml

1 M Sorbitol CaS

2 x 125 ml 2 x 60 ml

40% PEG CaT

25 ml 25 ml

Stab Vials: Pichia and E. coli stabs. Store at +4°C. Strain GS115 KM71 GS115 Albumin GS115 β-Gal TOP10F´Amount 1 stab 1 stab 1 stab 1 stab 1 stab Genotype his4 arg4 his4 aox1::ARG4 HIS4 HIS4 Phenotype (Pichia only) Mut + Mut S, Arg + Mut S Mut +

F´ {proAB, lacI q, lacZ∆M15, Tn10 (Tet R )} mcrA, ∆(mrr-hsdRMS-mcrBC), φ80lacZ∆M15, ∆lacX74, deoR, recA1, araD139, ∆(ara-leu)7697, galU, galK, rpsL(StrR), endA1, nupG λ-.

Box 2: Spheroplast Module. Store at -20°C. Reagent Zymolyase 1 M DTT Amount 10 x 20µl 10 x 1 ml Components 3 mg/ml Zymolyase in water (100,000 units/g lytic activity) 1 M dithiothreitol in water continued on next page

iii

Materials, continued
Kit Contents, continued
Box 3: Vectors and Primers. Store at -20°C Reagent pHIL-D2 10 µl (1 µg/µl) pPIC3.5 10 µl (1 µg/µl) pHIL-S1 10 µl (1 µg/µl) pPIC9 10 µl (1 µg/µl) 5´ AOX1 sequencing primer 20 µl (0.1 µg/µl, 15.6 pmoles/µl)3´ AOX1 sequencing primer 20 µl (0.1 µg/µl, 15.7 pmoles/µl) α-Factor sequencing primer 20 µl (0.1 µg/µl , 15.75 pmoles/µl) Description Vector for intracellular expression in Pichia Vector for intracellular expression in Pichia Vector for secreted expression in Pichia. Uses the PHO1 signal sequence Vector for secreted expression in Pichia. Uses the α-factor signal sequence 5´-GACTGGTTCCAATTGACAAGC-3´5´-GCAAATGGCATTCTGACATCC-3´ 5´-TACTATTGCCAGCATTGCTGC-3´

Media

The following prepackaged media is included for your convenience. Instructions for use are provided on the package. Media YP Base Medium YP Base Agar Medium Yeast Nitrogen Base Amount 2 pouches 2 pouches 1 pouch Yield 2 liters of YP medium 2 liters of YP medium 500 ml of 10X YNB

NOTE

The α-Factor, 5´ AOX1, and 3´ AOX1primers, and reagents to transform Pichia by spheroplasting (Pichia Spheroplast Module) are available separately from Invitrogen. Product Pichia Spheroplast Module 5´ AOX1 primer 3´ AOX1 primer 5´ and 3´ AOX1 primers α-Factor primer Reactions or Amount 10 spheroplast preparations (50 transformations) 2 µg, lyophilized 2 µg, lyophilized 2 µg, each, lyophilized 2 µg, each, lyophilized Catalog no.K1720-01 N710-02 N720-02 N740-02 N730-02 continued on next page

iv

Materials, continued
Required Equipment and Supplies (not provided)
• • • • • • • • • • • • • • • • 30°C rotary shaking incubator Water baths capable of 37°C, 45°C, and 100°C Centrifuge suitable for 50 ml conical tubes (floor or table-top) Baffled culture flasks with metal covers (50 ml, 250 ml, 500 ml, 1000 ml, and 3 L) 50 mlsterile, conical tubes 6 ml and 15 ml sterile snap-top tubes (Falcon 2059 or similar) UV Spectrophotometer Mini agarose gel apparatus and buffers Polyacrylamide Gel Electrophoresis apparatus and buffers Media for transformation, growth, screening, and expression (see Recipes, pages 53-58) 5% SDS solution (10 ml per transformation) Sterile cheesecloth or gauze Breaking Buffer (see Recipes, page...
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