All GP38 constructs were produced through de novo synthesis (GENEWIZ, Germantown, MD). tPA-GP38 strain IbAr 10200 (NC_005300) or strain Afg09-2990 (HM452306.1) was produced by the addition of the tPA secretion signal (MDAMKRGLCCVLLLCGAVFVSPS). Genes were cloned into the NotI and BglII sites of the pWRG7077 vector and verified by sequence analysis. For the histidine-tagged version of tPA-GP38 from strain IbAr 10200, six histidine residues were added to the C-terminal domain of the protein by de novo synthesis and cloned into the HindIII and XhoI site of pBFksr-HCacc-MCS, which contains a cytomegalovirus promotor (Biofactora).

The codon-optimized full-length M segment used was previously reported (25). The modified M segments lacking the mucin and/or GP38 regions were produced by polymerase chain reaction (PCR). ΔMUC was produced using the forward primer 5′-GATCTCCATCTTCAGGTTGTGGCTGCCGTGGGTCTC-3′ and reverse primer 3′-GAGACCCACGGCAGCCACAACCTGAAGATGGAGATC-5′, which removed the mucin-coding region in nucleotide regions 120 to 729. ΔMUCΔGP38 was produced using the forward primer 5′-ATCGCTGGGCTCCTCGCTGTGGCTGCCGTGGGTCTC-3′ and reverse primer 3′-GAGACCCACGGCAGCCACAGCGAGGAGCCCAGCGAT-5′, which removed nucleotide regions 120 to 1545. Both ΔMUC and ΔMUCΔGP38 constructs retained the signal sequences 1 to 117. All PCR reactions were performed using the Phusion polymerase (Invitrogen). Following PCR, fragments were digested with NotI and BglII and ligated into the pWRG7077 vector. Sequence analysis was used to verify that the changes had been successfully incorporated into the gene.

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