Ethical statement

Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (Assurance number A3381-01).

Statistics and reproducibility

No statistical method was used to predetermine sample size. Previous experience with the measurement techniques and their dynamic ranges was used to determine sample sizes. Large-scale experiments (i.e., Figs. 2 and 4) were not replicated in a separate experiment in their entirety, but the top conditions were independently replicated in separate experiments with matching results. Other experiments were generally replicated in a separate experiment at least once, with the results matching the data reported in this work. The experiments were not randomized because no animal studies or clinical trials were performed to assess a medical intervention. The Investigators were not blinded to allocation during experiments and outcome assessment because no animal studies or clinical trials were performed. One replicate of a control sample in the data related to Fig. 4 was excluded from analysis due to a liquid handling error. The replicate is included in the Source Data and annotated as excluded.

Samples were considered different from background if they exhibited p < 0.05 on a two-sided t-test and had a value that was beyond the limit of detection (average background ± 3x standard deviation of the background measurement). The Benjamini and Hochberg False Discovery Rate procedure⁵⁰ was used to correct for multiple testing. Statistical analyses were performed in python. Two-sided t-tests (two-tailed, two sample, assumed equal variance) were performed using the scipy package and the FDR procedure was performed using the statsmodels package with a family-wise error rate of 5%. For antibody screening, the following samples were used as a measurement of background, and the combined data were used in the t-test. Assembly: No DNA and beads only controls. S6P binding: No DNA and beads only controls. RBD binding: No DNA and beads only controls. ACE2 competition: No DNA and αHER2.

Pearson correlation coefficients were calculated using GraphPad Prism 9.

Software

Single-cell BCR sequences were analyzed with Cell Ranger v3.1.0. AlphaLISA data were analyzed with Prism 9.5.1. Images were processed using ImageJ2 v2.9.0/1.53t. Plate reader data were processed using Python 3.8.8. The Python code to analyze plate reader data was not central to the research and was not deposited.

Antibody sdFab sequence design

sdFabs were assembled based on a modified version of previously published protocols^16,17,18. Example plasmid maps of the aHER2 heavy and light chain sdFabs can be found in the Source data. Antibody sequences were collected from literature and their light chains were classified as either kappa or lambda via the terminal residue of the J-segment in the VL domain. The VH and VL domains were subsequently fused to their corresponding human constant heavy (Uniprot P0DOX5) or human constant light (kappa CL Uniprot P01834 or lambda 1 CL Uniprot P0CG04) chains. At the N-terminus of the VH and VL domains, we chose to include a modified expression tag based on the first 5-residues of the E. coli chloramphenicol acetyltransferase gene followed by a Tobacco Etch Virus (TEV) protease cleavage site (protein sequence: MEKKIENLYFQS, DNA sequence: atggagaaaaaaatcgaaaacctgtacttccagagc)⁷⁹ as opposed to the previously published SKIK tag⁸⁰. The heavy chain was fused to the LZA heterodimer subunit (AQLEKELQALEKENAQLEWELQALEKELAQK) and a strep II tag or super FLAG (sFLAG) tag⁸¹. The light chain was fused to the LZB heterodimer subunit (AQLKKKLQALKKKNAQLKWKLQALKKKLAQK). Antibodies in the screen in Fig. 2 were designed with the strep II tag on their heavy chain. Antibody sequences from the screens in Figs. 3 and 4 were designed with the sFLAG tag on their heavy chain. Antibodies in the screen in Fig. 2 and the EUA antibodies in Fig. 3 were designed with their native light chain class (kappa or lambda). Antibodies in the screen in Fig. 4 and the broadly neutralizing antibody sequences were designed with a kappa light chain, regardless of the native light chain class. Examples of the three types of antibody sequences are detailed below, with the important sequence features highlighted in square brackets [ ].

sdFab heavy chain constant strepII tagged:

[MEKKIENLYFQS][VH_Sequence][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC]GGGGS[AQLEKELQALEKENAQLEWELQALEKELAQK]GSSA[WSHPQFEK].

sdFab heavy chain constant super FLAG (sFLAG) tagged:

[MEKKIENLYFQS][VH_Sequence][ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC]GGGGS[AQLEKELQALEKENAQLEWELQALEKELAQK]GSSA[DYKDEDLL].

sdFab light chain kappa:

[MEKKIENLYFQS][VL_Sequence][RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]GGGGS[AQLKKKLQALKKKNAQLKWKLQALKKKLAQK].

sdFab light chain lambda 1:

[MEKKIENLYFQS][VL_Sequence][GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS]GGGGS[AQLKKKLQALKKKNAQLKWKLQALKKKLAQK].

DNA assembly and linear expression template (LET) generation

Proteins to be manufactured via CFPS were codon optimized using the IDT codon optimization tool and ordered as double-stranded linear DNA containing the desired Gibson assembly overhangs from IDT or GenScript. sfGFP was ordered containing the two pJL1 Gibson assembly overhangs. Antibody VH DNA was ordered with the pJL1 5′ and the human IgG1 heavy chain constant 5′ Gibson overhangs. Antibody VL DNA was ordered with the pJL1 5′ and the human Ig light chain kappa or lambda 1 Gibson assembly overhangs. DNA was resuspended at a concentration of 50 ng/μL and used without amplification.

Additional linear DNA components for Gibson assembly (pJL1 backbone, sdFab heavy chain constant strepII tagged, sdFab light chain kappa constant, sdFab light chain lambda 1 constant) were ordered as gblocks from IDT. These components were amplified using PCR using Q5 Hot Start DNA polymerase (NEB, M0493L) following manufacturer instructions. Amplified DNA was purified using the DNA Clean and Concentrate Kit (Zymo Research, D4006) and diluted to a concentration of 50 ng/μL. Sequences of the utilized components are listed below, with Gibson assembly sequences being denoted by underlined lowercase text and primers for a given amplicon being listed below the DNA sequence.

Gibson assembly overhangs:

pJL1 5′ Gibson: tttgtttaactttaagaaggagatatacat.

pJL1 3′ Gibson: gtcgaccggctgctaacaaagcccgaaagg.

Human IgG1 heavy chain constant 5′ Gibson: gcgtcaacaaaaggtccttcagttttcccattagcccct.

Human Ig light chain kappa 5′ Gibson: cgcacggtcgcggcgccgtctgtctttatttttcctcct.

Human Ig light chain lambda 5′ Gibson: ggccaacccaaagcaaacccaactgtcactttgttcccg.

Linear pJL1 plasmid backbone (Addgene plasmid # 69496):

gtcgaccggctgctaacaaagcccgaaaggAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGCCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCTTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATtttgtttaactttaagaaggagatatacat.

pJL1_F: gtcgaccggctgcta.

pJL1_R: atgtatatctccttcttaaagttaaacaaaattatttcta.

Linear sdFab heavy chain constant strepII tagged:

gcgtcaacaaaaggtccttcagttttcccattagcccctTCTTCTAAGTCAACTAGTGGCGGTACTGCCGCTCTTGGGTGTTTGGTTAAAGATTACTTCCCAGAACCGGTTACGGTCTCGTGGAACTCTGGTGCACTGACATCGGGCGTACATACATTTCCCGCAGTTTTGCAGTCTTCGGGACTGTATTCTCTTTCATCGGTGGTTACAGTCCCTAGCTCTTCCCTGGGTACACAGACCTACATTTGTAATGTTAATCATAAGCCGAGTAATACTAAGGTGGATAAAAAGGTGGAACCGAAGTCTTGTGGTGGTGGCGGGTCAGCTCAACTGGAGAAGGAGTTACAGGCACTGGAAAAAGAGAATGCTCAACTTGAGTGGGAATTACAGGCGTTAGAAAAAGAACTGGCCCAGAAGGGTTCTAGCGCATGGTCACATCCCCAGTTCGAAAAATAAgtcgaccggctgctaacaaagcccgaaagg.

Linear sdFab heavy chain constant super FLAG tagged:

gcgtcaacaaaaggtccttcagttttcccattagcccctTCTTCTAAGTCAACTAGTGGCGGTACTGCCGCTCTTGGGTGTTTGGTTAAAGATTACTTCCCAGAACCGGTTACGGTCTCGTGGAACTCTGGTGCACTGACATCGGGCGTACATACATTTCCCGCAGTTTTGCAGTCTTCGGGACTGTATTCTCTTTCATCGGTGGTTACAGTCCCTAGCTCTTCCCTGGGTACACAGACCTACATTTGTAATGTTAATCATAAGCCGAGTAATACTAAGGTGGATAAAAAGGTGGAACCGAAGTCTTGTGGTGGTGGCGGGTCAGCTCAACTGGAGAAGGAGTTACAGGCACTGGAAAAAGAGAATGCTCAACTTGAGTGGGAATTACAGGCGTTAGAAAAAGAACTGGCCCAGAAGGGTGGAGCCAGTCCAGCAGCTCCTGCGCCTGGCGGGGACTACAAAGATGAAGACCTTCTTTAAgtcgaccggctgctaacaaagcccgaaagg.

IgGC_F: GCGTCAACAAAAGGTCCTTCAGTTTTC.

pJL1_3′Gib_R: CCTTTCGGGCTTTGTTAGCAGC.

Linear sdFab light chain kappa constant:

cgcacggtcgcggcgccgtctgtctttatttttcctcctTCTGATGAACAGCTTAAATCTGGGACAGCTTCTGTTGTATGTTTATTAAACAACTTTTACCCGCGTGAGGCAAAAGTTCAATGGAAGGTAGACAACGCACTGCAAAGCGGAAATTCGCAGGAGTCAGTTACCGAACAGGATTCCAAGGATAGTACCTACTCCTTAAGTTCAACATTAACCCTGTCAAAGGCGGACTATGAAAAACATAAGGTATATGCCTGCGAAGTAACTCATCAGGGCTTATCATCCCCAGTTACAAAATCTTTCAACCGTGGAGAATGCGGCGGCGGAGGTAGCGCGCAGCTTAAGAAAAAATTGCAAGCCCTTAAAAAAAAAAATGCCCAACTTAAATGGAAGCTGCAAGCCTTAAAAAAGAAATTGGCGCAGAAGTAAgtcgaccggctgctaacaaagcccgaaagg.

kLC_F: TCGCGGCGCCGTCTG.

pJL1_3′Gib_R: CCTTTCGGGCTTTGTTAGCAGC.

Linear sdFab light chain lambda 1 constant:

ggccaacccaaagcaaacccaactgtcactttgttcccgCCCTCAAGCGAGGAACTTCAGGCTAATAAGGCCACGCTTGTTTGCCTGATCTCAGACTTTTATCCCGGTGCCGTAACAGTGGCTTGGAAGGCAGATGGTTCGCCGGTCAAAGCGGGCGTGGAAACTACAAAGCCATCGAAACAGTCAAACAATAAATATGCGGCATCAAGTTACTTGAGCCTTACCCCAGAACAGTGGAAGTCACACCGCTCGTACAGTTGTCAAGTTACACACGAGGGAAGTACAGTTGAAAAGACCGTTGCCCCAACTGAATGTTCAGGCGGTGGTGGCTCAGCGCAGTTAAAGAAAAAACTGCAGGCTTTGAAGAAAAAGAATGCTCAATTAAAGTGGAAATTGCAGGCGTTGAAGAAGAAACTTGCGCAGAAGTAAgtcgaccggctgctaacaaagcccgaaagg.

lLC_F: GGCCAACCCAAAGCAAACC.

pJL1_3′Gib_R: CCTTTCGGGCTTTGTTAGCAGC.

Linear sfGFP (same DNA sequence as Addgene Plasmid #102634). Note that the sequence of sfGFP is heavily modified and contains mutations from Bundy et al.⁸².

tttgtttaactttaagaaggagatatacatATGAGCAAAGGTGAAGAACTGTTTACCGGCGTTGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGTCACAAATTCAGCGTGCGTGGTGAAGGTGAAGGCGATGCCACGATTGGCAAACTGACGCTGAAATTTATCTGCACCACCGGCAAACTGCCGGTGCCGTGGCCGACGCTGGTGACCACCCTGACCTATGGCGTTCAGTGTTTTAGTCGCTATCCGGATCACATGAAACGTCACGATTTCTTTAAATCTGCAATGCCGGAAGGCTATGTGCAGGAACGTACGATTAGCTTTAAAGATGATGGCAAATATAAAACGCGCGCCGTTGTGAAATTTGAAGGCGATACCCTGGTGAACCGCATTGAACTGAAAGGCACGGATTTTAAAGAAGATGGCAATATCCTGGGCCATAAACTGGAATACAACTTTAATAGCCATAATGTTTATATTACGGCGGATAAACAGAAAAATGGCATCAAAGCGAATTTTACCGTTCGCCATAACGTTGAAGATGGCAGTGTGCAGCTGGCAGATCATTATCAGCAGAATACCCCGATTGGTGATGGTCCGGTGCTGCTGCCGGATAATCATTATCTGAGCACGCAGACCGTTCTGTCTAAAGATCCGAACGAAAAAGGCACGCGGGACCACATGGTTCTGCACGAATATGTGAATGCGGCAGGTATTACGTGGAGCCATCCGCAGTTCGAAAAATAAgtcgaccggctgctaacaaagcccgaaagg.

Gibson assembly was used to assemble protein open reading frame DNA with the pJL1 backbone following the published protocol with the addition of 3.125 μg/mL of ET SSB (NEB, product no. M2401S)^83,84. 20 ng of purified, linear pJL1 backbone, 20 ng of purified, linear sdFab VH or VL constant DNA, and 20 ng of the protein open reading frame insert were combined in 2 μL Gibson assembly reactions and incubated at 50 °C for 30 min. The unpurified assembly reactions were diluted in 40 μL of nuclease-free water (Fisher Scientific, AM9937) and 1 μL of the diluted reaction was used as the template for a PCR to generate linear expression templates (LETs) for CFPS. Linear expression templates were amplified via PCR using the pJL1_LET_F (ctgagatacctacagcgtgagc) and pJL1_LET_R (cgtcactcatggtgatttctcacttg) primers in a 50 μL PCR reaction using the Q5 Hot Start DNA polymerase (NEB, M0493L) following manufacturer instructions.

The DNA sequence of the P. pyralis luciferase containing a c-terminal strepII tag (fLuc, Uniprot Q27758) used as a negative control is below and was cloned into the pJL1 vector.

atggaagacgctaagaacattaagaagggacctgctccattctaccccctcgaagacggcactgcaggtgagcagcttcataaagcgatgaagcgttatgcgttagttcctggcacgatcgccttcactgacgcgcacatcgaagtcaatatcacctacgctgaatactttgagatgagtgtgcgtctggcggaagccatgaagcgttatggccttaacacgaaccaccgcatcgttgtttgtagcgagaattccttacaattcttcatgcccgtccttggcgcgctgtttattggtgtggccgttgcaccagccaatgacatctataatgagcgcgagttgttgaactccatgaacatttctcaaccaacagtggtgttcgtttcaaagaaaggcttacagaaaatcttaaacgttcaaaagaaactgccgattatccagaagatcatcattatggatagtaagactgactaccagggcttccagtcaatgtatacattcgtgacgagtcacctgcccccgggttttaacgagtacgactttgtcccagagagctttgatcgcgacaagaccatcgccctcattatgaatagcagtggttcgacgggtagcccaaagggagtggccctgccccatcgtaccgcgtgcgtccgtttctcccatgcccgcgacccaattttcggcaatcaaatcatccccgacacggcaatcttgtcggtcgtcccgtttcaccatggctttggaatgtttacgacactcggttacctcatctgcggtttccgcgtcgttctgatgtatcgcttcgaggaagagttgttcttacgttcgcttcaggactacaagattcaatccgcccttctggtccccactttgttcagtttctttgctaagagcaccttaattgataagtatgacctctccaacttacacgagattgcgagcggtggtgctcccctcagcaaagaggttggagaggcggttgctaagcgttttcatctgcccggtatccgtcaaggttacggcctcaccgaaaccacttctgccattcttatcactccggaaggtgacgataagcctggggcagtgggtaaagttgtacccttcttcgaggctaaggttgtggatttagatacggggaagaccttaggtgtgaaccagcgcggtgaactgtgcgttcgcggtccgatgattatgtcgggttatgttaatgaccccgaggctacgaacgcgcttatcgataaggacggttggcttcattccggcgacatcgcttactgggatgaggatgagcacttcttcatcgttgaccgtctgaagagtctcatcaagtataagggatgtcaagtcgctccggcagagttagagagcatcttactccagcaccctaatatcttcgatgctggggttgccgggctcccaggcgacgatgccggcgagctgccggcggcggtagttgttttagagcatggcaagaccatgaccgaaaaggagattgtagactacgtcgcgagtcaagtaaccacagcgaagaagctccgcggtggagtggtctttgttgacgaggtgcctaaaggcctgacgggcaaacttgacgcgcgtaagatccgtgagatcctcatcaaagcgaagaagggtgggaagagtaagctggggagttcaggttggtcccacccgcaatttgagaagtga.

Cell extract preparation for cell-free protein synthesis

E. coli Origami^TM B(DE3) (Novagen, 70837) extracts were prepared using a modified version of established protocols^85,86. Briefly, a 150 mL Origami^TM B(DE3) starter culture was inoculated in LB from a glycerol stock and cultured in a 250 mL baffled flask at 37 °C for 16 h. The 2xYTP was prepared without glucose in 75% of the final volume and sterilized using an autoclave. A 4x glucose solution was prepared and autoclaved separately, then added to the medium immediately before use. The starter cultures were used to inoculate 1 L of 2xYTPG media (16 g/L tryptone, 10 g/L yeast extract, 5 g/L sodium chloride, 7 g/L potassium phosphate dibasic, 3 g/L potassium phosphate monobasic, 18 g/L glucose) in a 2.5 L Full-Baffle Tunair shake flask at an initial OD600 of 0.08. Cells were cultured at 37 °C at 220 RPM in a shaking incubator. Cultures were grown until OD600 0.4-0.6, at which point the expression of T7 RNA polymerase was induced by the addition of IPTG to a final concentration of 0.5 mM. Cells were harvested at an OD600 of 2.5 via centrifugation at 12,000 × g for 1 min at 4 °C. Cell pellets were washed three times with 25 mL S30 buffer per 50 mL culture (10 mM Tris Acetate pH 8.2, 14 mM Magnesium Acetate, and 60 mM Potassium Acetate). Pellets were resuspended in 1 mL S30 buffer per gram of cell mass. Cell suspensions were lysed using a single pass on an Avestin EmulsiFlex-B15 Homogenizer at a lysis pressure of 24,000 PSI. Cell debris was separated via centrifugation at 18,000 × g for 20 min, and the clarified lysate was collected, flash-frozen in liquid nitrogen, and stored at −80 °C.

Cell-free protein synthesis reactions

CFPS reactions were composed of the following reagents: 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 1.2 mM ATP, 0.5 mM of each CTP, GTP, and UTP. 0.03 mg/mL folinic acid, 0.17 mg/mL E. coli MRE600 tRNA (Roche 10109541001), 100 mM NAD, 50 mM CoA, 4 mM oxalic acid, 1 mM putrescine, 1 mM spermidine, 57 mM HEPES pH 7.2, 2 mM of each amino acid, 33.3 mM PEP, 20% v/v E. coli extract, varying concentrations of DNA template, and the remainder water. The preparation of these reagents has been described in detail elsewhere⁸⁷. For DNA templates, plasmids were used at a concentration of 8 nM, and unpurified linear PCR products were used at 6.66% (v/v). For the expression of antibodies, each template was added to a final concentration of 6.66% (v/v). For antibody and sdFab expression 4 mM oxidized glutathione, 1 mM reduced glutathione, 14 μM of purified DsbC, and 50 μM FkpA were also supplemented to the reactions. In addition, for oxidizing CFPS reactions, cell-extracts were treated with 500 μM iodoacetamide (IAM) at room temperature for 30 min before use in CFPS⁸⁸. All reaction components were assembled on ice and were either run as 12 μL reactions in 1.5 mL microtubes or 2 μL reactions in 384-well plates (BioRad, HSP3801). For 2 μL reactions, components were transferred to the plate using an Echo 525 acoustic liquid handler. A mix containing all the CFPS components except for the DNA was dispensed from 384PP Plus plates (Labcyte, PPL-0200) using the BP setting. The DNA (unpurified PCR products) was dispensed from a 384LDV Plus plate (Labcyte, LPL-0200) using the GP setting. Reactions were allowed to proceed at 30 °C for 20 h.

Quantification of cell-free protein synthesis reactions

To quantify sfGFP fluorescence, a standard curve was prepared using previously reported methods⁸⁶. Radioactive leucine was added to CFPS at a final concentration of 10 μM of L-[14C(U)]-leucine (Perkin Elmer NEC279E250UC, 11.1GBq/mMole), followed by precipitation of the expressed proteins and scintillation counting⁸⁹. To quantify sfGFP fluorescence, 2 μL of a CFPS reaction was diluted in 48 μL of water in a Black Costar 96 Well Half Area Plate. Fluorescence was measured using a BioTek Synergy^TM H1 plate reader with excitation and emission wavelengths of 485 and 528, respectively. Scintillation counts and fluorescence were fit to determine a standard curve for use with non-radioactive samples.

To visualize antibody assembly, proteins were labeled during CFPS with FluoroTect^TM (Promega, L5001). FluoroTect^TM was included in the CFPS reaction at 3.33%v/v. After protein synthesis, RNAseA (Omega Bio-Tek, AC118) was added to 0.1 mg/mL and the sample was incubated at 37 °C for 10 min. 3 μL of the CFPS and RNAseA mixture were combined with 4x loading buffer (LiCor, 928-40004) and the samples were subsequently denatured at 70 °C for 3 min, then separated via SDS-PAGE and imaged using a LI-COR Odyssey Fc imager on the 600 channel. Densitometry was performed using the ImageJ software.

DsbC and FkpA expression and purification

Protein expression, purification, and his tag removal were performed similarly to previously reported⁷⁷. DsbC (Uniprot P0AEG6, residues 21–236) and FkpA (Uniprot P45523, residues 26–270) were ordered as gBlocks from IDT containing a c-terminal, TEV cleavable his tag (GSENLYFQSGSHHHHHHHHHH) and cloned into pET28a. Plasmid maps of both DsbC and FkpA are available in the Source Data. Plasmids were transformed into BL21 Star^TM DE3, plated on LB agar, and cultured overnight at 37 °C. 1 L of Overnight Express TB (Fisher Scientific, 71491-4) was inoculated by scraping all colonies on a transformation plate and cultured at 37 °C in 2.5 L tunair flasks (IBI Scientific, SS-8003) at 220 rpm overnight. Cells were harvested, resuspended at a ratio of 1 g cell mass to 4 mL resuspension buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 1X HALT protease inhibitor without EDTA (Fisher Scientific, 78429), 1 mg/mL lysozyme, 62.5 U/mL cell suspension of benzonase (Sigma-Aldrich, E1014-25KU)) and lysed using an Avestin B15 homogenizer at 24,000 PSI. The lysate was spun down 14,000 × g for 10 min and the clarified supernatant was incubated with Ni-NTA Agarose (Qiagen, 30230) for 60 min on an end-over-end shaker. The resin was spun down 2500l × g for 2 min, the supernatant removed, resuspended in wash buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 50 mM Imidazole), loaded on a gravity flow column, and subsequently washed with 20X resin volumes of wash buffer. Protein was eluted using elution buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 500 mM Imidazole) and exchanged into 50 mM HEPES pH 7.4, 150 mM NaCl using PD-10 desalting columns (Cytvia, 17-0851-01) according to manufacturer instructions.

His tags were removed via cleavage by ProTEV Plus (Promega, V6102). Before cleavage, 10% v/v glycerol was added to the protein. ProTEV Plus was added to a concentration of 0.5 U/μg purified protein and DTT was added to a concentration of 1 mM. Cleavage reactions were carried out at 30 °C for 4 h. Free His tag and ProTEV Plus were removed by incubating with Ni-NTA Agarose for 1 h at 4 °C and collecting the supernatant. Proteins were subsequently concentrated to >1 mg/mL (Millipore, UFC800396). His tag removal was validated via SDS-PAGE and the AlphaScreen Histidine (Nickel Chelate) Detection Kit (Perkin Elmer, 6760619C).

AlphaLISA reactions

AlphaLISA reactions were carried out in 50 mM HEPES pH 7.4, 150 mM NaCl, 1 mg/mL BSA, and 0.00015 v/v TritonX-100 (hereafter referred to as Alpha buffer). All components were dispensed using an Echo 525 liquid handler from a 384-Well Polypropylene 2.0 Plus microplate (Labcyte, PPL-0200) using the 384PP_Plus_GPSA fluid type. All components of the AlphaLISA reactions were prepared as 4x stocks and added as 0.5 μL to the final 2 μL reaction to achieve the desired concentration. All AlphaLISA reactions were performed with CFPS reactions diluted to a final concentration of 0.025 v/v. AlphaLISA beads were combined to prepare a 4X stock in Alpha buffer immediately before use and added to the proteins to yield a concentration of 0.08 mg/mL donor beads and 0.02 mg/mL acceptor beads in the final reaction. All reactions were incubated with AlphaLISA beads for at least 1 h before measurement. AlphaLISA measurements were taken on a Tecan Infinite M1000 Pro plate reader using the AlphaLISA filter with an excitation time of 100 ms, an integration time of 300 ms, and a settling time of 20 ms. Before measurement, plates were allowed to equilibrate inside the instrument for 10 min. For measurements involving sdFabs, protein A AlphaLISA beads were avoided due to the ability of protein A to bind human subgroup VH3 Fabs⁹⁰.

The impact of CFPS reagents on AlphaLISA was determined by serially diluting the specified reagents in Alpha buffer and combining them with the specified AlphaLISA conditions. The TrueHits kit (Perkin Elmer, AL900) was used to assess the impact of the CFPS reagents on the Alpha detection chemistry. CFPS reagents were mixed with the donor and acceptor beads and incubated for 2 h before measurement. His tagged RBD (Sino Biological, 40592-V08H) and human FC tagged human ACE2 (GenScript, Z03484) were used to evaluate the impact of CFPS reagents on capture chemistries. RBD and ACE2 were diluted in Alpha buffer, mixed at a final reaction concentration of 10 nM each, combined with the CFPS reagents, and allowed to incubate for 1 h. Donor and acceptor beads were subsequently added and allowed to incubate for a further hour before measurement. Protein A Alpha donor beads (Perkin Elmer, AS102), Ni-Chelate AlphaLISA acceptor beads (Perkin Elmer, AL108), and anti-6xhis AlphaLISA acceptor beads (Perkin Elmer, AL178) were utilized for detection.

The commercial neutralizing antibody ACE2 competition experiment was performed with the following antibodies: nAb1 (Acro Biosystems, SAD-S35), nAb2 (Sino Biological, 40592-MM57), nAb3 (Sino Biological, 40591-MM43), nAb4 (Sino Biological, 40592-R001). ELISA IC₅₀ values were recorded from the product page at the time of purchase and converted to μg/mL assuming a MW of 150,000 Da if reported in M. Antibodies were serially diluted in Alpha buffer and mixed with SARS-CoV-2 RBD (Sino Biological, 40592-V02H) at a concentration of 10 nM in the final reaction and incubated for 1 h. Mouse FC tagged human ACE2 (Sino Biological, 10108-H05H) was subsequently added and incubated for 1 h, followed by simultaneous addition of the acceptor and donor beads. AlphaLISA detection was performed using Anti-Mouse IgG Alpha Donor beads (PerkinElmer, AS104) and Strep-Tactin AlphaLISA Acceptor beads (PerkinElmer, AL136). IC₅₀ values were calculated using Prism 9 by fitting the normalized data to [Inhibitor] vs. response–Variable slope (four parameters) fit with the max constrained to a value of 1.

For all antibody screening experiments, the different reagents and AlphaLISA conditions used are described in Supplementary Table 2. The different AlphaLISA measurements were carried out as described below.

Assembly AlphaLISA reactions consisted of sdFab expressing CFPS and either Rabbit Anti-Human kappa light chain antibody (Abcam, ab125919) or Rabbit Anti-Human lambda light chain (Abcam, ab124719). CFPS reaction containing the expressed sdFab of interest was mixed with the appropriate anti-light chain antibody and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.

SARS-CoV-2 S6P binding AlphaLISA reactions consisted of sdFab expressing CFPS and SARS-CoV-2 S6P CFPS reaction containing the expressed sdFab of interest was mixed with the S6P and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.

SARS-CoV-2 RBD binding AlphaLISA reactions consisted of sdFab expressing CFPS and SARS-CoV-2 RBD. CFPS reaction containing the expressed sdFab of interest was mixed with the RBD and allowed to equilibrate for two hours before the simultaneous addition of the acceptor and donor beads.

ACE2 and RBD competition AlphaLISA reactions consisted of sdFab expressing CFPS, human ACE2, and SARS-CoV-2 S6P. CFPS reaction containing the expressed sdFab of interest was first mixed with S6P and allowed to incubate for 1 h. Subsequently, ACE2 was added and allowed to equilibrate for a further 1 h before the simultaneous addition of the acceptor and donor beads.

For SARS-CoV-2 variant and other non-SARS-CoV-2 coronavirus binding experiments, AlphaLISA measurements were carried out in the same manner as described for SARS-CoV-2 S6P. The following Hisx6-tagged proteins were used. SARS-CoV-2 S6P (Acro Biosystems, SPN-C52H9), SARS-CoV-2 S6P Alpha/ B.1.1.7 (Gift from Lauren Carter at the Institute for Protein Design at the University of Washington, expressed and purified as described elsewhere⁷⁷), SARS-CoV-2 S6P Beta/B.1.351 (Gift from Lauren Carter at the Institute for Protein Design at the University of Washington, expressed and purified as described elsewhere⁷⁷), SARS-CoV-2 S6P Gamma/P.1 (Gift from Lauren Carter at the Institute for Protein Design at the University of Washington, expressed and purified as described elsewhere⁷⁷), SARS-CoV-2 S6P Delta/B.1.617.2 (AcroBiosystems, SPN-C52He), SARS-CoV-2 S6P Omicron/BA.1 (AcroBiosystems, SPN-C52Hz), SARS-CoV-2 S6P Omicron/BA.2 (AcroBiosystems, SPN-C5223), SARS-CoV-2 S6P Omicron/BA.2.12.1 (AcroBiosystems, SPN-C522d), SARS-CoV-2 S6P Omicron/BA.4/5 (AcroBiosystems, SPN-C522e), SARS-CoV S2P (AcroBiosystems, SPN-S52H6), MERS-CoV S2P (AcroBiosystems, SPN-M52H4), HCoV-HKU1 S (AcroBiosystems, SPN-H52H5), HCoV-OC43 S (AcroBiosystems, SPN-H52Hz), HCoV-NL63 S (AcroBiosystems, SPN-H52H4), and HCoV-229E S (AcroBiosystems, SPN-H52H3).

In the dose-dependent ACE2 competition titration experiments CFPS reactions were incubated with SARS-CoV-2 RBD for 1 h followed by the addition of the specified concentration (two-fold serially diluted from 100 nM) of human ACE2. All three components were incubated for an additional hour prior to simultaneous addition of AlphaLISA beads. Reactions were incubated for 2 h prior to measurement.

For RBD and ACE2 bridging experiments SARS-CoV-2 RBD, human ACE2, and the specified dilution of CFPS (two-fold serially diluted from 0.025 v/v) were incubated for 1 h prior to the simultaneous addition of the AlphaLISA beads. Reactions were incubated for 2 h prior to measurement.

Mouse Immunization, cell staining, and sorting

Female C57BL/6 (Strain: 000664) were purchased from The Jackson Laboratory. Six-week-old animals were immunized with 10¹⁰ viral particles (vp) of ChAd-SARS-CoV-2-S⁷³ in 50 µl of PBS via intramuscular injection in the hind leg. Draining inguinal lymph nodes were collected 10 days later and processed into a single-cell suspension. Cells were stained with biotinylated recombinant SARS-CoV-2 spike (S2P) for 30 min at 4 °C then washed twice with FACS buffer followed by staining with anti-CD19 BV421 (BioLegend # 115537), anti-CD4 FITC (BioLegend # 100405), anti-IgD-PE-Cy7 (BioLegend # 405719), Streptavidin APC (BioLegend # 405207), aqua cell viability dye (Invitrogen L34957), and anti-mouse CD16/CD32 Fc block (BioLegend # 156607). Spike-positive activated B cells (live singlet CD4- CD19+ IgDlo Streptavidin+) were bulk sorted on BD FACSAriaII sorter.

Single-cell RNA-seq library preparation and sequencing

The following 10x Genomics kits were used for libraries preparation: Chromium Single Cell 5′ Library and Gel Bead Kit v2 (PN-1000006), Chromium Single Cell A Chip Kit (PN-1000152), Chromium Single Cell V(D)J Enrichment Kit, Mouse B cell (96rxns) (PN-1000072), and Single Index Kit T (PN-1000213). The GEM generation and barcoding was followed by cDNA preparation then GEM RT reaction and bead cleanup steps. Purified cDNA was amplified for 10–14 cycles then cleaned up using SPRIselect beads. cDNA concentration was determined by running samples on a Bioanalyzer. BCR target enrichments were done on the full-length cDNA followed by BCR libraries preparation as recommended by 10x Genomics Chromium Single Cell V(D)J Reagent Kits (v1 Chemistry) user guide. The cDNA Libraries were sequenced on Novaseq S4 (Illumina), targeting a median sequencing depth of 5000 read pairs per cell.

Processing of single-cell BCR sequences

Demultiplexed pair-end FASTQ reads from 10x Genomics single-cell V(D)J profiling were preprocessed using the “cellranger vdj” command from Cell Ranger v3.1.0 for alignment against the GRCm38 mouse reference v3.1.0 (refdata-cellranger-vdj-GRCm38-alts-ensembl-3.1.0), generating 3760 assembled high-confidence BCR sequences for 4420 cells. Sequences for screening were selected randomly from the top clonal groups with >10 members in the clonal group. Cellranger vdj output was then parsed using Change-O v0.4.6 within the immcantation suite. Additional quality control included examining sequences to be productively rearranged and have valid V and J gene annotations. Furthermore, only cells with exactly one heavy chain sequence paired with at least one light chain sequence were kept.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-023-38965-w