Part:BBa_K1983006

gp45 phenylalanine mutant M37 with C-terminal 6XHis-Tag

T4 phage clamp protein gp45 with 37 stable mutations to phenylalanine.

Overview

The Gp45 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 five mutants of Gp45 ranging from 6 to 37 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 4,8% in the wild type protein to 21,2% of phenylalanine in Gp45m37 protein.

Table 1.: Comparison of phenylalanine quantities between the wild type and the mutant Gp45 proteins.

This version of mutant has 37 mutations to phenylalanine, resulting in a total of 21,2%.

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 NdeI and XhoI. The fragments containing mutant genes were cloned into pET21b expression vector digested with the same restriction enzymes. Transformant colonies were PCR-screened using Prom7/Term7 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 C41 strains, with BL21 (DE3) strain showing the best results (fig. 2).

Figure 2. 12% SDS-PAGE analysis of modified Gp45 protein expression levels. E. coli BL21 (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 (Csm4M11, Csm4M42 and Gp45M37) of the family 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 XhoI site found at 688
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]