Part:BBa_K3580200

Alanine racemase with 6xHis-Tag

BBa_K3580200 is an AR sequence with 6xHis tag for affinity purification method widely established with minimal impact on the activity and structure of the target protein.

This part includes the same sequence of alr as BBa_K1172901, but it is new having 6xHis sequence. 6xHis has the same amino acid sequence with BBa_K1223006 but some codons are different. We expressed alanine racemase from this parts with T5.

Expression and purification of alanine racemase

In our project, we inserted this part between bacteriophage T5 promoter for E. coli RNA polymerase and lambda t0 terminator of pCA24N(-gfp) planmid. This plasmid was transformed into the BL21(DE3) star strain. As this vector is chloramphenicol resistant, we always cultured the bacteria in media containing chloramphenicol. After expression induction by IPTG, the BL21 cells were collected by centrifugation, then sonicated and His-Tag purification was performed. (wikiマテメソハイパーリンク) The His-Tag purified Alr was confirmed with SDS-PAGE(Fig.1). The meanings of each abbreviation are given in Table 1. The concentration of purified racemase was quantified with the Bradford method.

Fig.1 SDS-PAGEs of purified E. coli BL21(DE3)star containing BBa_K3580200

Table.1 Each meaning of abbriviation

We decided to assay AR by using PURE frex provided by GeneFrontier, Inc. As PURE frex does not contain any extra metabolic enzymes other than those involved in transcription and translation, we would be able to perform the assays without considering the problems of controlling the metabolic system. (wikiマテメソハイパーリンク) 

Consequently, to assay the activity of purified AR, the racemization reaction was performed with the following solution(Table.2) and incubated at 37°C for 1 h. The sample was Filtered using centrifugal filter device at 14,000g for 30min (Amicon Ultra-0.5 Centrifugal Filter Devices 3K) and the filtered sample was collected. The filter device was washed prior to centrifugation of the sample using the buffer used in the AR reaction. the sample was centrifuged to collect the filtered product. Filtering allowed us to collect substances other than AR from the incubated product. Fluorescence values of GFPS1 synthesized by the combination of PURE frex containing 19 L-amino acids and a plasmid encoding the reporter protein GFPS1 with L-alanine, D-alanine, and the reaction solution filtration products of AR and D-alanine (presumably containing PLP, potassium phosphate, and racemic alanine) were measured(Fig.2).

Fig.2 Effect of L-alanine substrate repletion for translation by racemase

The results demonstrated that GFP could not be synthesized by D-alanine alone, but L-alanine produced by racemase-mediated racemization of D-alanine and L-alanine could be used for translation, creating a situation in which fluorescence was restored by the synthesized GFP.  

Table.2  Alr racemization reaction assay solution 

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 367
  Illegal NotI site found at 1121
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 291
  Illegal BamHI site found at 21
  Illegal BamHI site found at 993
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 409
  Illegal AgeI site found at 709
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 166