Title:Characterization of recombinant glutathione reductase from the psychrophilic Antarctic bacterium Colwellia psychrerythraea

Authors: Mikyoung Ji1, Callie V. Barnwell2, Amy M. Grunden1*

1Department of Plant and Microbial Biology

4550A Thomas Hall Campus Box 7612

North Carolina State University

Raleigh, NC 27695 USA

2Department of Animal Science

123 Polk Hall Campus Box 7621
North Carolina State University
Raleigh, NC 27695 USA

*Corresponding Author: Amy M. Grunden

Email Address:

Phone number: (919)513-4295

Online Resource 1The sequences and features of primers used in construction of the glutathione reductase mutations in E. coli strains BL21 (λDE) and JM105

E.coli gor
(outer
region) / Forward
5’-TGGTCAGCGGGATAGCAGAAGG-3’ / outer region of the gene
(-388 to -366)
Reverse
5’-CAGTGGATGAGTAAGCGGATGC-3’ / outer region of the gene
(+166 to +188)
PCR
product
(gor-Cm
specific)
used in
BL21(DE) and JM105
strain / Forward
5’-CGCTACAATCGCGGTAATCAACGATAAGG
ACACTTTGTCATGTAGGCTGGAGCTGCTTCG-3’ / *Homologous
region of
gene
*Chloramphenicol
gene cassette
region
Reverse
5’-TTTTGGTTGCCCCCATCTTCAGCGCCACCGC
GAAGCCCTGCATATGAATATCCTCCTTAG-3’

Online Resource 2 Specific activities and yields of recombinant C. psychrerythraeaglutathione

reductase at each purification step

Purification
Step / Protein Amount
(mg) / Specific Activity (unit/mg)a / % of yield
Cell Extract / 908.55 / 15.14 / 100
DEAE colume / 146.64 / 112.14 / 16.14
Phenyl Sepharose / 64 / 141.95 / 7.04
Blue Sepharose / 26.6 / 380.42 / 2.93

a One unit stands for reduction of 1.0 µmole of oxidized glutathione per minute at pH 7.6 at 25oC

Online Resource 3 Estimation of the molecular weight of recombinant C. psychrerythraea glutathione reductase using gel filtration

Online Resource 4Two-way Analysis of Variance (ANOVA) table with two fixed effects, strains,

and diamide exposure at three different temperatures.

18oC / 25oC / 37oC
Source / DF / SS / MS / F / P value / DF / SS / MS / F / P value / DF / SS / MS / F / P value
Strain / 2 / 0.480 / 0.240 / 87.92 / <.000 / 2 / 0.432 / 0.216 / 31.68 / <.000 / 2 / 0.162 / 0.081 / 9.16 / 0.002
Diamide / 2 / 0.497 / 0.248 / 90.94 / <.000 / 2 / 1.312 / 0.656 / 96.14 / <.000 / 2 / 0.621 / 0.310 / 34.99 / <.000
Strain*
Diamide (interaction) / 4 / 0.171 / 0.043 / 15.68 / .
<.000 / 4 / 0.157 / 0.039 / 5.767 / 0.003 / 4 / 0.041 / 0.010 / 1.16 / 0.362

Online Resource 5 Results of Student’s two tail t test with equal distribution

Growth Temperature / Tested strains with diamide treatment / P value
37 oC / MLJ701 (0 mM) vs MLJ70 (0 mM) / 0.2306
25 oC / MLJ701 (0 mM) vs MLJ70 (0 mM) / 0.3912
18 oC / MLJ701 (0 mM) vs MLJ70 (0 mM) / 0.0097
37 oC / MLJ701 (0.2 mM) vs MLJ702 (0.2 mM) / 0.0955
37 oC / MLJ701 (0.3 mM) vs MLJ702 (0.3 mM) / 0.1330
25 oC / MLJ701 (0.2 mM) vs MLJ702 (0.2 mM) / 0.0014
25 oC / MLJ701 (0.3 mM) vs MLJ702 (0.3 mM) / 0.0053
18 oC / MLJ701 (0.2 mM) vs MLJ702 (0.2 mM) / 0.0007
18 oC / MLJ701 (0.3 mM) vs MLJ702 (0.3 mM) / 0.0003
18 oC / MLJ702 (0 mM) vs MLJ702 (0.2 mM) / 0.7131
18 oC / MLJ702 (0 mM) vs MLJ702 (0.3 mM) / 0.0035
18 oC / MLJ701 (0 mM) vs MLJ701 (0.2mM) / 0.0024
18 oC / MLJ701 (0 mM) vs MLJ701 (0.3mM) / 0.0013
18 oC / MLJ702 (0.2 mM) vs MLJ70 (0.2 mM) / 0.0136
18 oC / MLJ702 (0.3 mM) vs MLJ70 (0.3 mM) / 0.0014
37 oC / MLJ701 (0.2 mM) vs MLJ70 (0.2 mM) / 0.0454
37 oC / MLJ701 (0.3 mM) vs MLJ70 (0.3 mM) / 0.0415
25 oC / MLJ701 (0.2 mM) vs MLJ70 (0.2 mM) / 0.0014
25 oC / MLJ701 (0.3 mM) vs MLJ70 (0.3 mM) / 0.0057
18 oC / MLJ701 (0.2 mM) vs MLJ70 (0.2 mM) / 0.0086
18 oC / MLJ701 (0.3 mM) vs MLJ70 (0.3 mM) / 0.0111