Artificial Extracellular Matrix Proteins Containing Phenylalanine Analogues Biosynthesized in Bacteria Using T7 Expression System and the PEGylation

Abstract

In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC).

Click grafting of alkyne-containing vinyl polymers onto biosynthesized extracellular matrix protein containing azide functionality and adhesion control of human umbilical vein endothelial cells

“…We have already reported the in vivo translational activities of the Phe analogues, ( p -N 3 Phe, p -BrPhe, p -IPhe, p -ethynylphenylalanine, p -cyanophenylalanine, and p -AcPhe). 11 M9 minimal medium (1 L) supplemented with 0.4 % glucose, 35 mg/L thiamine, 0.1 mM MgSO 4 , 0.1 mM CaCl 2 , the 20 naturally occurring amino acids (at 40 mg/L each), and antibiotics (kanamycin 25 mg/L, chloramphenicol 20 mg/L) was inoculated with 300 mL of culture containing the expression strain. When the optical density at 600 nm reached 0.7–1.0, a medium shift was performed.…”

“…Either when fibronectin coats a plate or is present in the extracellular matrix, interaction with HUVEC are mediated by integrin receptors such as α5β1, 7 and α4β1. 8,11 The sequence motif REDV, found within the CS5 domain, is thought to be the shortest sequence capable of α4β1 integrin binding. 8b In Tirrell’s studies on aECM-CS5-ELF, 6c,6e HUVEC adhesion and spreading were dependent of the presence of the CS5 domain of fibronectin.…”

“…For example, incorporation of phenylalanine (Phe) analogues [including p -N 3 Phe, p -bromophenylalanine ( p -BrPhe), and p -iodophenylalanine ( p -IPhe) having an orthogonal functional group] into a recombinant protein enables chemical modification via “click” reactions, Heck reactions, or Sonogashira couplings, and has been accomplished using a bacterial expression host that harbors a mutated phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. 10,11 Tirrell et al previously identified mutations which enlarge the Phe binding cavity of E. coli PheRS, Ala294Gly 10a and Thr251Gly, 10b and produced the corresponding Ala294Gly (PheRS*) 10a and Thr251Gly/Ala294Gly 10b (PheRS**) mutants, respectively. The Thr251Gly/Ala294Gly double mutant, PheRS**, effectively incorporates even p -acetylphenylalanine ( p -AcPhe) into mouse dihydrofolate reductase when the T5 system is used.…”

“…10b Although the amount of material produced is insufficient due to the moderate strength of the T5 promoter, we previously reported that this limitation can be overcome using a combination of T7 promoter and T7 RNA polymerase. 11 The T7 RNA polymerase has greater activity and is more selective than the endogenous E. coli RNA polymerase. 12–14 The T7 expression system (pET system) is capable of producing large amounts of protein (up to 100 mg/L of culture medium).…”

“…17 In the present study, biosynthesized aECM-CS5-ELF containing p -N 3 Phe (aECM-CS5-ELF-N 3 ) was prepared using PheRS** and a T7 promoter. 11 Next, alkyne-containing trithiocarbonate was generated for use as the RAFT agent for controlled radical polymerization of three vinyl monomers: styrene, acrylamide, and N -isopropylacrylamide. Hybridization of the aECM with vinyl polymers is accomplished via a copper-catalyzed alkyne-azide click reaction 18 based on a “ grafting onto ” procedure (Figure 1).…”