. 2018 Jun 27;8:9725. doi: 10.1038/s41598-018-27860-w

Table 3.

Immunological detection of NylC-antigenic protein and RT-PCR analysis.

Mutant enzymes derived from NylC_p2	Mutant phenotype*¹	Western blotting*²		Direct ELISA*³ (µg ml⁻¹)	RT-PCR*⁴ (%)
Mutant enzymes derived from NylC_p2	Mutant phenotype*¹	Soluble	Insoluble	Direct ELISA*³ (µg ml⁻¹)	RT-PCR*⁴ (%)
P¹²²	Type 4	—	—	8.2	129
H¹²²	Type 4	—	—	2.4	142
W¹²²	Type 4	—	—	3.2	112
G¹²²Y¹³⁰A³⁶Q²⁶³	Type 1	27 kDa, 9 kDa	—	11.4	100
G¹²²Y¹³⁰A³⁶Q²⁶³-T¹⁷⁹	Type 4	—	—	8.1	121
G¹²²Y¹³⁰A³⁶Q²⁶³-T²⁹⁵	Type 1	27 kDa, 9 kDa	—	17.7	113
G¹²²Y¹³⁰A³⁶Q²⁶³-D²⁹⁹	Type 4	—	—	4.4	154
G¹²²Y¹³⁰A³⁶Q²⁶³-E²⁹⁹	Type 3	—	36 kDa	11.7	106
G¹²²Y¹³⁰A³⁶Q²⁶³-V⁷⁵	Type 3	—	36 kDa	1.1	120
G¹²²Y¹³⁰A³⁶Q²⁶³-R¹⁰⁶	Type 3	—	36 kDa	0.2	118
G¹²²Y¹³⁰A³⁶Q²⁶³-L¹⁴¹	Type 4	—	—	2.9	124
G¹²²Y¹³⁰A³⁶Q²⁶³-L¹⁵⁷	Type 3	—	36 kDa	3.1	96
G¹²²Y¹³⁰A³⁶Q²⁶³-N¹⁹¹	Type 1	27 kDa, 9 kDa		14.3	121
G¹²²Y¹³⁰A³⁶Q²⁶³-D²³⁵	Type 3	—	36 kDa	6.9	67

^*1NylC mutants were classified into four types (see Fig. 2b). Type 1: obtained as active enzymes, Type 2: obtained as soluble precursors (e.g., NylC_p2-A¹³⁷; see Table 1). Type 3: obtained as insoluble precursors (protein aggregation). Type 4: subjected to fragmentation during the cultivation/purification process.

^*2The cell extract (soluble fraction) and precipitates obtained by the centrifugation of sonicated cells (insoluble fraction) were boiled in SDS, and NylC-antigenic proteins were detected by Western blot analysis using anti-NylC antibody (Fig. S4).

^*3The amount of NylC-antigenic protein was analyzed by direct ELISA using anti-NylC antibody. The antigenic protein was estimated by subtracting the background level (4.9 µg ml⁻¹ for cell extracts harboring the vector pBluescript) from the data obtained for E. coli clones harboring the mutant nylC gene.

^*4To check the expression of the mutant nylC gene, we prepared RNA samples from E. coli clones. Reverse transcription (RT)-PCR analysis revealed that PCR products corresponding to nylC-mRNA were present in all mutants.