Increased antibody expression from Escherichia coli through wobble-base library mutagenesis by enzymatic inverse PCR

• Published in: Gene Vol. 123; no. 1; pp. 1 - 7
• Main Authors: Stemmer, Willem P.C., Morris, Suzanne K., Kautzer, Curtis R., Wilson, Barry S.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 15.01.1993; Amsterdam Elsevier; New York, NY
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites, Antibody - genetics; Binding Sites, Antibody - immunology; Biological and medical sciences; Biotechnology; class-IIS restriction enzymes; Cloning, Molecular - methods; DNA, Recombinant; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Library; Genetic engineering; Genetic technics; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - immunology; Indium - immunology; LEIPCR; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; mRNA folding; Mutagenesis; Nucleic Acid Conformation; Polymerase Chain Reaction - methods; protein production level; Protein Sorting Signals - genetics; Recombinant DNA; Restriction Mapping; ribosome binding; RNA, Messenger - chemistry; screening; signal peptide; screening; ribosome binding; protein production level; nt; mRNA folding; dNTP; class-IIS restriction enzymes; Recombinant DNA; bp; Ap; Tc; LEIPCR; kb; oligo; ENase; wt; aa; RBS; SDS; PAGE; mAb; signal peptide; Fv; cfu; PolIk; EIPCR; IPTG; PCR; Screening; Leader peptide; Antibody; heavy-Peptide chain; Escherichia coli; Site directed mutagenesis; Production; Bacteria; Fv-Fragment; Gene expression; Vector; Enterobacteriaceae
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(93)90531-7

We tested the value of a new library mutagenesis approach, called library enzymatic inverse PCR (LEIPCR), for expression-level enhancement of antibody Fv fragments produced in Escherichia coli. The production level of active, metal chelate-specific antibody from our constructs is limited by a low expression level of the second, heavy-chain cistron. To increase the production level, LEIPCR was applied to the wobble bases of the second cistron leader peptide. In LEIPCR mutagenesis, the entire plasmid is amplified using mutagenic primers with class-IIS restriction endonuclease (ENase) sites at their 5′ ends. The PCR product is digested with the class-IIS ENase (here, BsaI; GGTCTCN ▾NNN ▴), which removes its own recognition sequence, and the ends are self-ligated. Thus, LEIPCR can be used to make plasmid mutant libraries regardless of the nucleotide sequence, and independent of available ENase sites. The resulting library of 10 7 wobble mutants was screened for active Fv by a colony filter lift. A selected mutant was shown to produce between four- and elevenfold more active Fv than the wild type (wt), and fivefold more heavy chain. Mutations outside of the leader peptide were shown not to be involved. The mutated areas of the mRNAs of two different up-mutants may have less secondary structure than the wt. Thus, the sequence of the mRNA of the second leader peptide was limiting to the expression level of heavy-chain and active Fv.