IPTG诱导的原核表达(英文)[文].pdfVIP

IPTG 诱导的原核表达 Chapter 15, Protocol 1 Expression of Cloned Genes in E. coli Using IPTG-inducible Promoters This protocol describes how (1) to clone cloned This protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying IPTG-inducible promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. .CAUTION .RECIPE . MA TERIALS Buffers and Solutions Coomassie Brilliant Blue stain or Silver stain. IPTG (1 M). 1x SDS gel-loading buffer. . Nucleic Acids and Oligonucleotides Gene or cDNA fragment of interest . Media LB agar plates containing 50 μg/ml ampicillin. LB medium containing 50 μg/ml ampicillin. . Additional Reagents Step 1 of this protocol requires the reagents listed in Chapter 8, Protocol 7. Step 2 of this protocol requires the reagents listed in Chapter 1, Protocol 17 or Chapter 1, Protocol 19. Step 3 of this protocol requires the reagents listed in Chapter 1, Protocol 23 to Chapter 1, Protocol 26. Step 4 of this protocol requires the reagents listed in Chapter 12, Protocol 3. . Vectors and Bacterial Strains E. coli strain suitable for transformation and carrying either the lacIq or lacIq1 allele Some IPTG-inducible expression vectors carry the lacIq allele on the expression plasmid (e.g., pMAL and pGEX). These can be used in any laboratory strain of E. coli (e.g., JM101, DH5F, and TG1). IPTG-inducible expression vector Other examples include pGEM -3Z (Promega), pGEX-1 (Pharmacia), pKK223-3 (Pharmacia), pMEX (U.S. Biochemicals), pTrc 99A (Pharmacia), and pMAL (New England Biolabs). Positive control plasmid (e.g., an IPTG-inducible vector known to express a LacZ fusion protein of defined size) . . METHOD 1..Modify by PCR (Chapter 8, Protocol 7), or isolate by restriction enz