Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA.

The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

Combinatorial assembly of clone libraries using site-specific recombination.

Generation of DNA clones for use in proteomic and genomic research often requires a significant level of parallel production, as the number of downstream options for these experiments increases. Where a single fluorescently tagged construct may have sufficed before, there is now the need for multiple types of labels for different readouts and different assays. Protein expression, which once utilized a very small set of vectors because of low throughput expression and purification, has now rapidly matured into a high throughput system in which dozens of conditions can be tested in parallel to identify the best candidate clones. This has returned the bottleneck in many of these technologies to the generation of DNA clones, and standard cloning techniques often dramatically limit the throughput and success of such processes. In order to overcome this bottleneck, higher-throughput and more parallel cloning processes need to be developed which would allow rapid, inexpensive production of final clones. In addition, there is a strong need to utilize standardized elements to avoid unnecessarily remaking fragments of clones that could be used in multiple constructs. The advent of recombinational cloning helped to increase the parallel processing of DNA clones, but was still limited by the need to generate different vector backbones for each specific need. The solution to this problem emerged with the introduction of combinatorial approaches to clone construction, based on either homologous or site-specific recombination processes. In particular, the Gateway Multisite system provides all of the necessary components for a highly parallel, inexpensive, rapid, and diverse platform for clone construction in many areas of proteomic and genomic research. Here we describe our optimized system for combinatorial cloning, including improvements in cloning protocols and construct design that permit users to easily generate libraries of clones which can be combined in parallel to create an unlimited number of final constructs. The system is capable of utilizing the tens of thousands of commercially available Gateway clones already in existence, and allows easy adaptation of most DNA vectors to the system.

Characterization of recombinant human IL-15 deamidation and its practical elimination through substitution of asparagine 77.

PURPOSE: The use of recombinant human interleukin (rhIL)-15 as a potential therapeutic immune modulator and anticancer agent requires pure, stable preparations. However, purified rhIL-15 preparations readily accumulated heterogeneities. We sought to improve rhIL-15 stability through process, formulation, and targeted amino acid changes. METHODS: The solution state of rhIL-15 versus buffer composition and temperature was studied using SEC and IEX methods. rhIL-15 deamidation was confirmed using RP-HPLC/ESI-MS, enzymatic labeling, and peptide mapping. Deamidation kinetics were measured versus buffer composition and pH using RP-HPLC. Deamidation-resistant rhIL-15 variants (N77A, N77S, N77Q, G78A, and [N71S/N72A/N77A]) were produced in E. coli, then assayed for T-cell culture expansion potency and deamidation resistance. RESULTS: Adding 20% ethanol to buffers or heating at ≥32°C dispersed rhIL-15 transient pairs, improving purification efficiencies. Asparagine 77 deamidated rapidly at pH 7.4 with activation energy of 22.9 kcal per mol. Deamidation in citrate buffer was 17-fold slower at pH 5.9 than at pH 7.4. Amino acid substitutions at N77 or G78 slowed deamidation ≥23-fold. rhIL-15 variants N77A and (N71S/N72A/N77A) were active in a CTLL-2 proliferation assay equivalent to unsubstituted rhIL-15. CONCLUSIONS: The N77A and (N71S/N72A/N77A) rhIL-15 variants are resistant to deamidation and remain potent, thus providing enhanced drug substances for clinical evaluation.