The Directed Evolution, High-level Expression, Single-step Purification And The Application Of Escherichia Coli Alkaline Phosphatase

Directed evolution was used to enhance both catalytic activity and expression level of Escherichia coli alkaline phosphatase. Through one round of error-prone PCR, followed by a rapid and sensitive screening procedure, one improved variant from about 4000 mutant colonies was obtained and sequenced. Seven mutant sites located on the whole phoA gene, four sites on the regulating gene and the others on the structure gene. To analysis their different influences in the protein expression and catalysis activity, single site mutations'and combined site mutations'variants were constructed respectively. The variant with a single-point mutation in -10 TATA conserved region of promoter sequence showed a six folds expression level above the wide type has been quantified by real-time PCR of phoA gene mRNA. Quantification showed that wide expression range difference does correlate with the transcprit numbers variance. The reason for the variant with one mutation between the SD sequence and the transcription start site how to increase expression two-fold is not clear. Probable reason may due to GC-rich sequences or unstable mRNA secondary structure. Another mutant with the mutation lead to enhanced SD sequence owe to higher complementarity to the E.coli 16S rRNA sequence, which enhanced translation efficiencies, has three-fold expression level above the wild type. The combined mutation variants with the three sites mentioned above showed accumulative effect on the protein expression level. The silent mutation on the phoA signal peptide did not correlate with the higher expression level. Three activating amino acid substitutions, Q30L, K328Q, and T441A were identified by sequencing the structure gene. The Q30L and T441A variants, which mutations were located far away from the center of the catalytic pocket, showed little variance with the wide type. The transphosphorylation activity of the K328Q enzyme was selectively enhanced, while the hydrolysis activity was reduced compared to that of the wild-type enzyme, in comparison with the wild-type enzyme. Several other mutant enzymes share this characterictic of the K328Q enzyme. These results suggests that the positive charge at position 328 is at least partially responsible for mainting the balance between the hydrolysis and transphosphorylation activities and plays an important role in determining the rate-limiting step of E.coli alkaline phosphatase. The mutated Escherichia coli phoA structural gene, which encodes an EAP 4-186 with increased specific activity, was cloned into pASK75 expression vector fused to OmpA signal peptide and under the transcriptional control of tetA promoter. The EAP-linker-Strep-tag II fusion protein was expressed under different Tc concentration, temperature, and induced for different time. Among these tests, when the cell incubated with high concentration of Tc most of the secreted EAP formed inclusion bodies in the periplasm, However, EAP could be produced as a soluble form without the reduction of expression level by inducing with less Tc. Furthermore, reducing the incubation temperature to 28 ℃instead of 37 ℃will improve the expression level by EAP activity determination. Up to 5.5 g/l EAP fusion protein can be obtained from Escherichia coli under these optimal conditions. Furthermore, EAP-linker-Strep-tagII fusion protein can be fast and simply purified through Strep-Tactin mix. Fusion protein displays turnover and spectroscopic properties that are essentially to those of nature enzyme. As a result, pASK75 vector was successfully used to obtain high expression level and efficiency purification of EAP. This paper describes a generally, applicable method using Streptavidin ligand Strep-tag II for fast purification and oriented immobilization of EAP onto Strep-Tactin coated surface. Orientation of EAP resulted in higher residual activity compared to the random immobilized EAP through physical adsorption.The coding sequence of three streptavidin affinity peptides with different affinity constants Strep-tag I, Strep-tag II and SBP-tag were fusioned with the C-termius of EAP coding sequence. The three kinds of fuion proteins: EAP 4-186-linker-SBP-tag,EAP 4-186-linker-Strep-tag I and EAP 4-186-linker-Strep-tag II interact with the streptavidin coated on the 96 wells, different color was obtained after adding the EAP substrate pNPP due to the different binding capacity of three kinds of affinity tag toward Strep-Tactin. Combing thespecific binding character of this attachment, this oriented method can be extended to a preparatory mode including high-throughput screening assays for high-affinity binding tag using EAP as molecular signals.