**4. Materials and Methods**

#### *4.1. Bioinformatics Analysis for DNA Sequences of P. americana Thymosin*

We identified the gene and transcript sequences of *P. americana* thymosin from genomic and transcriptional databases which were built by our lab [49]. The introns and exons of the *P. americana* thymosin gene sequence were analyzed by the softberry program (http://linux1.softberry.com/all.htm). According to alternative splicing, there are three different transcript variants (THY1, THY2, and THY3) in *P. americana* thymosin. All complete thymosin cDNA sequences were deposited in GenBank (accession No. MK573540, MK573541, MK573542). The DNA sequences were analyzed by the online program at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Conserved motifs were determined using Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif\_scan) and signal peptide was predicted using the SignalP Server (http://www.cbs.dtu.dk/services/SignalP/). The theoretical PI and molecular mass were estimated by ExPASy (http://www.expasy.ch/tools/peptide-mass.html).

#### *4.2. Cloning, Expression, and Purification of Pa-THYs*

The sequences of THYs were cloned into a PET-28(a) vector, the proteins Pa-THYs were expressed in *E. coli* cells and purified by His Trap TMFF crude. Briefly, the sequences of THYs were amplified from the total cDNA of *P. americana* with specific primers, as follows: PET-Pa-F (*EcoR I*): 5-*CGC*GAATTCATGTCGGCCCCAGTC-3; PET-Pa-R (*Xho I*): 5-CCGCTCGAGTTATGCTTTCTT CTCTTCATCG-3 and then cloned into PET-28(a) vector at *EcoR I* and *Xho I* recognition sites. The plasmids of PET-THY1, PET-THY2, and PET-THY3 were transformed into *E. coli* BL21 (DE3) competent cells for the final expression. The proteins (Pa-THY1, Pa-THY2, and Pa-THY3) were induced by isopropyl β-D-thiogalactoside (IPTG) at 37 ◦C for 6 h. The cells were harvested by centrifuging at 8000 rpm for 10 min and suspending in a 1× PBS phosphate buffer. The *E. coli* was shattered using ultrasonication, then centrifuged at 12,000× *g* for 10 min at 4 ◦C to remove the precipitate. The recombined proteins were purified by His Trap TMFF crude (1 mL) according to the manufacturer's instructions and analyzed with SDS-PAGE. To ge<sup>t</sup> the high concentration of these proteins, they were concentrated by an ultrafiltration centrifuge tube (Millipore, Massachussettes, USA), and the total

protein concentration was measured by BCA (bicinchoninic acid) protein assay kit (Biosharp, Hefei, Anhui, China) according to the manufacturer's instructions.
