Part:BBa_K1983009

Streptococcus thermofilus Csm4 phenylalanine mutant M42 with C-terminal 6XHis-Tag

Csm4 phenylalanine mutant M42 with C-terminal 6XHis-Tag

Overview

The Csm4 phenyalanine mutants belong to PolyPhe protein family used to condense free phenylalanine from the outer medium. PolyPhe proteins are distinctive for their high rates of phenylalanine. This protein coding biobrick part among with other twins was created to assess the bacteria‘s capability to synthesize phenylalanine-rich proteins. There are three mutants of Csm4 ranging from 11 to 42 mutations changing five canonical aromatic or hydrophobic amino acids (Tyr, Trp, Leu, Ile, Met) to phenylalanine (see design page for further information). The whole range spans from 7,7% in the first mutant to 21% of phenylalanine in the third mutant (Table 1.).

Table 1.: Comparison of phenylalanine quantities between the wild type and the mutant Csm4 proteins.

This version of mutant has 42 mutations to phenylalanine, resulting in a total of 21%.

Figure 1. First graph: the red line represents dG of the wild type protein, and the red dot represents Nmut having the same dG as the wild type protein. Second graph: the red dots represent mutation minimal extremities that destabilize protein the least.

Experiments and Results

Cloning

The received sequences were amplified using Uni-FW/RV primers and digested with Esp3I and XhoI. The fragments containing mutant genes were cloned into pETDuet™ expression vector digested with NcoI and XhoI. Transformant colonies were PCR-screened using Up-1A/Down-3 primers and positive clone plasmids were sequenced prior to further usage.

Expression assays

The gp45 mutant protein expression was tested in Escherichia coli BL21; ER2566 and TOP10 strains, with ER2566 strain showing the best results (fig. 2).

Figure 2. 12% SDS-PAGE analysis of modified Csm4 protein expression levels. E. coli ER2566 (DE3) was induced by IPTG (2 µM final concentration) using pET expression system. Red star indicated the weight of target protein. Odd numbers represent soluble protein fraction, even numbers represent insoluble fraction.

Characterization in vivo

Synthetic PolyPhe proteins are enriched in phenylalanine and, therefore, a cell has to use more of this amino acid to produce the protein. As a result, cell’s pool of phenylalanine rapidly decreases. Consequently, a target amino acid from extracellular environment is absorbed in order to increase inner phenylalanine pool and continue translation of PolyPhe protein. As a result, cells demonstrate higher consumption of phenylalanine and its concentration outer environment decreases faster than normally. We have characterized three protein mutants of the family (Csm4M11, Csm4M42 and Gp45M37) by evaluating the uptake of phenylalanine from the special substrate medium (fig. 3).

Figure 3. Cell’s activity in reduction of 1.1 g of phenylalanine in growth medium (pH 7.4, 37ºC) while expressing PolyPhe over 30-minute period. Control is cells without any biobrick. Gp45 (37) was expressed in E. coli BL21, Csm4 (11) and Csm4 (42) in E. coli ER2566. Results are achieved by 2.5 grams of cells.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 796
  Illegal XhoI site found at 907
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]